Prenylation inhibitors and methods of their synthesis and use

ABSTRACT

The present invention is directed to compounds useful in the treatment of diseases associated with prenylation of proteins and pharmaceutically acceptable salts thereof, to pharmaceutical compositions comprising same, and to methods for inhibiting protein prenylation in an organism using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/636,327, filed Aug. 6, 2003, now U.S. Pat. No. 7,166,619, which is acontinuation-in-part of U.S. patent application Ser. No. 10/336,285, nowU.S. Pat. No. 6,649,638, filed Jan. 3, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/219,628,filed Aug. 14, 2002, now abandoned. Each of these priority documents isincorporated herein, in its entirety, by reference.

FIELD OF THE INVENTION

This present invention relates to a class of novel compounds useful inthe treatment of diseases associated with the prenylation of proteins.

BACKGROUND OF THE INVENTION

The mammalian Ras proteins are a family of guanosine triphosphate (GTP)binding and hydrolyzing proteins that regulate cell growth anddifferentiation. Their overproduction or mutation can lead touncontrolled cell growth, and has been implicated as a cause oraggravating factor in a variety of diseases including cancer,restenosis, psoriasis, endometriosis, atherosclerosis, viral or yeastinfection, and corneal neovascularization.

Ras proteins share characteristic C-terminal sequences termed the CAAXmotif, wherein C is Cys, A is an amino acid, usually an aliphatic aminoacid, and X is an aliphatic amino acid or other type of amino acid. Thebiological activity of the proteins is dependent upon thepost-translational modification of these sequences by isoprenoid lipids.For proteins having a C-terminal CAAX sequence, this modification, whichis called prenylation, occurs in at least three steps: the addition ofeither a 15 carbon (farnesyl) or 20 carbon (geranylgeranyl) isoprenoidto the Cys residue, the proteolytic cleavage of the last three aminoacids from the C-terminus, and the methylation of the new C-terminalcarboxylate. Zhang and Casey, Ann. Rev. Biochem. 1996, 65, 241-269. Theprenylation of some proteins may include a fourth step; thepalmitoylation of one or two Cys residues N-terminal to the farnesylatedCys.

Ras-like proteins terminating with XXCC or XCXC motifs can also beprenylated and are modified by geranylgeranylation on the Cys residues.These proteins do not require an endoproteolytic processing step. Whilesome mammalian cell proteins terminating in XCXC are carboxymethylated,it is not clear whether carboxymethylation follows prenylation ofproteins terminating with XXCC motifs. Clarke, Ann. Rev. Biochem., 1992,61, 355-386. For all Ras-like proteins, however, addition of theisoprenoid is the first step of prenylation, and is required for thesubsequent steps. Cox and Der, Critical Rev. Oncogenesis, 1992, 3,365-400; and Ashby et al., Curr. Opinion Lipidology, 1998, 9, 99-102.

Three enzymes have been found to catalyze protein prenylation:farnesyl-protein transferase (FPTase), geranylgeranyl-proteintransferase type I (GGPTase-I), and geranylgeranyl-protein transferasetype-II (GGPTase-II, also called Rab GGPTase). These enzymes are presentin both yeast and mammalian cells. Schafer and Rine, Annu. Rev. Genet.,1992, 30, 209-237. U.S. Pat. No. 5,578,477 discloses a method ofpurifying FPTase using recombinant technology and yeast host cells. Suchtechniques are useful in the elucidation of the enzyme structures.

FPTase and GGPTase-I are α/β heterodimeric enzymes that share a common αsubunit; the β subunits are distinct but share approximately 30% aminoacid identity. Brown and Goldstein, Nature, 1993, 366, 14-15; Zhang etal., J. Biol. Chem., 1994, 269, 3175-3180. GGPTase II has different αand β subunits, and complexes with a third component (REP, Rab EscortProtein) that presents the protein substrate to the α/β catalyticsubunits. GGPTase proteins, and the nucleic acid sequence encoding them,are disclosed by U.S. Pat. No. 5,789,558 and WO 95/20651. U.S. Pat. No.5,141,851 discloses the structure of a FPTase protein.

Each of these enzymes selectively uses farnesyl diphosphate orgeranylgeranyl diphosphate as the isoprenoid donor, and selectivelyrecognizes the protein substrate. FPTase farnesylates CAAX-containingproteins that end with Ser, Met, Cys, Gln or Ala. GGPTase-Igeranylgeranylates CAAX-containing proteins that end with Leu or Phe.For FPTase and GGPTase-I, CAAX tetrapeptides comprise the minimum regionrequired for interaction of the protein substrate with the enzyme.GGPTase-II modifies XXCC and XCXC proteins, but its interaction withprotein substrates is more complex, requiring protein sequences inaddition to the C-terminal amino acids for recognition. Enzymologicalcharacterization of FPTase, GGPTase-I and GGPTase-II has demonstratedthat it is possible to selectively inhibit only one of these enzymes.Moores e al., J. Biol Chem., 1991, 266, 17438.

GGPTase-I transfers a geranylgeranyl group from the prenyl donorgeranylgeranyl diphosphate to the cysteine residue of substrate CAAXprotein. Clarke, Annu. Rev. Biochem., 1992, 61, 355-386; Newman andMagee, Biochim. Biophys. Acta, 1993, 1155, 79-96. Known targets ofGGPTase-I include the gamma subunits of brain heterotrimeric G proteinsand Ras-related small GTP-binding proteins such as RhoA, RhoB, RhoC,CDC42Hs, Rac1, Rac2, Rap1A and Rap1B. The proteins RhoA, RhoB, RhoC,Rac1, Rac2 and CDC42Hs have roles in the regulation of cell shape.Ridley and Hall, Cell, 1992, 70, 389-399; Ridley et al, Cell, 1992, 70,401-410; Bokoch and Der, FASEB J., 1993, 7, 750-759. Rac and Rapproteins play roles in neutrophil activation.

It has been found that the ability of Ras proteins to affect cell shapeis dependant upon Rho and Rac protein function. See, e.g., Mackey andHall, J. Biol. Chem., 1998, 273, 20688-20695. It thus follows thatbecause Rho and Rac proteins require geranylgeranylation for function,an inhibitor of GGPTase-I would block the functions of these proteins,and may be useful as, for example, an anticancer agent. This notion issupported by recently reported research.

For example, GGPTase-I inhibitors can arrest human tumor cells that lackp53 in G0/G1, and induce the accumulation of p21^(WAP). This suggeststhat these inhibitors could be used to restore growth arrest in cellslacking functional p53. Vogt et al., J. Biol. Chem., 1997, 272,27224-27229. Noteworthy in this regard are reports indicating thatK-Ras, the form of Ras gene most associated with human cancers, can bemodified by GGPTase-I in cells where FPTase is inhibited. Whyte et al.,J. Biol. Chem., 1997, 272, 14459-14464. Since geranylgeranylated Ras hasbeen reported to be as efficient as the farnesylated form in celltransformation studies, K-Ras cancers could be treated with GGPTase-Iinhibitors. Lerner et al., J. Biol. Chem., 1995, 270, 26770-26773.

In addition to cancer, there are other pathological conditions for whichGGPTase inhibitors may be used as intervention agents. These include,for example, the intimal hyperplasia associated with restenosis andatherosclerosis. Pulmonary artery smooth muscle cells seem particularlysensitive to inhibition of GGPTase-I, and treatment of such cells with aGGPTase inhibitor resulted in a superinduction of their induciblenitric-oxide synthase (NOS-2) by interleukin-1β. Finder et al., J. Biol.Chem., 1997, 272, 13484-13488.

GGPTase inhibitors may also be used as anti-fungal agents. In S.cerievisiae and Candida albicans, and apparently most other fungi, cellwall biosynthesis is controlled by a Rho-type protein that is modifiedby the fungal GGPTase-I. Qadota et al., Science, 1996, 272, 279-281.Selective inhibition of the fungal enzyme would diminish cell wallintegrity, and thus be lethal to fungal cells.

Numerous other prenylation inhibitors have been studied. Some examplesof these are disclosed by U.S. Pat. Nos. 5,420,245; 5,574,025;5,523,430; 5,602,098; 5,631,401; 5,705,686; 5,238,922; 5,470,832; and6,191,147; and by European Application Nos. 856,315 and 537,008. Theeffectiveness and specificity of these inhibitors vary widely, as dotheir chemical structures, and many of them are difficult to synthesizeand purify.

Therefore, there is a need for new, more effective prenyl-proteintransferase inhibitors.

SUMMARY OF THE INVENTION

The present invention provides a group of structurally-related compoundsdisclosed below that are effective as inhibitors of protein prenylation.

The present invention also provides a composition comprising a compoundof the present invention, or a pharmaceutically-acceptable salt thereof,and a pharmaceutically-acceptable carrier.

The present invention also provides a method for inhibiting proteinprenylation comprising contacting an isoprenoid transferase with acompound of the present invention or a pharmaceutically-acceptable saltthereof. As used herein, an “isoprenoid transferase” refers to anyenzyme capable of transferring an isoprenoid group, for example,farnesyl or geranylgeranyl, to a protein, e.g., Ras or Ras-likeproteins. Such isoprenoid transferases include FPTase, GGPTase I andGGPTase II. Unless the context requires otherwise, the term “contacting”refers to providing conditions to bring the compound into proximity toan isoprenoid transferase to allow for inhibition of activity of theisoprenoid transferase. For example, contacting a compound of thepresent invention with an isoprenoid transferase can be accomplished byadministering the compound to an organism, or by isolating cells, e.g.,cells in bone marrow, and admixing the cells with the compound underconditions sufficient for the compound to diffuse into or be activelytaken up by the cells, in vitro or ex vivo, into the cell interior. Whenex vivo administration of the compound is used, for example, in treatingleukemia, the treated cells can then be reinfused into the organism fromwhich they were taken.

Such method for inhibiting protein prenylation can be used, for example,in prevention and/or treatment of a disease or condition in a plant oranimal that is caused, aggravated or prolonged by Ras or Ras-likeprotein prenylation. In animals, such diseases include, but are notlimited to, cancer, restenosis, psoriasis, endometriosis,atherosclerosis, ischemia, myocardial ischemic disorders such asmyocardial infarction, high serum cholesterol levels, viral infection,fungal infections, yeast infections, bacteria and protozoa infections,and disorders related to abnormal angiogenesis including, but notlimited to, corneal neovascularization. In plants, such diseases includeyeast and viral infections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general synthetic approach for the production ofprenylation inhibitors of the present invention having a centralpyrazole ring.

FIG. 2 illustrates a specific embodiment of the general syntheticapproach for the production of prenylation inhibitors shown in FIG. 1.

FIG. 3 illustrates a synthetic scheme for the production of an ethergroup that links a central aromatic ring having two heteroatoms to anamide group that may be further substituted to form prenylationinhibitor compounds of the present invention.

FIG. 4 illustrates a synthetic scheme for the production of a phenylgroup that links a central aromatic ring having two heteroatoms to anamide group that may be further substituted to form prenylationinhibitor compounds of the present invention.

FIG. 5 illustrates a synthetic scheme for the production of an aminegroup that links a central aromatic ring having two heteroatoms to anamide group that may be further substituted to form prenylationinhibitor compounds of the present invention.

FIG. 6 illustrates a synthetic scheme for making substitutions at the4-position of a 5-membered aromatic ring having two heteroatoms withinprenylation inhibitor structures of the present invention.

FIG. 7 illustrates the formation of a compound of the present inventionhaving a central pyrazole ring structure.

FIG. 8 a illustrates the synthesis of alkyl linking groups withincompounds of the present invention through an ethyl-methylamine linkinggroup.

FIG. 8 b illustrates the synthesis of a compound of the presentinvention having a phenyl linking group added to a pyrazole ring.

FIG. 9 illustrates the synthesis of prenylation inhibitors of thepresent invention with amine linking groups having different alkylsubstituents.

FIG. 10 illustrates the synthesis of prenylation inhibitors of thepresent invention having a central pyrazole ring with a substitutedalkyl group.

FIG. 11 illustrates the synthesis of prenylation inhibitors of thepresent invention having an ether linker on a central pyrazole ring.

FIG. 12 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central phenyl ring to which a phenyl linkinggroup is attached.

FIG. 13 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central phenyl ring attached to an etherlinking group.

FIG. 14 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central pyrimidine ring matched with a phenyllinking group.

FIG. 15 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central pyrimidine ring with an ether linkinggroup.

FIG. 16 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central oxazole ring and a phenyl linkinggroup.

FIG. 17 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central pyrazole ring and a thiophene linkinggroup.

FIG. 18 illustrates the synthesis of a prenylation inhibitor of thepresent invention having a central pyrazole ring arid an amine linkinggroup.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “organism” includes plants and animals.Exemplary animals include mammals, fish, birds, insects, and arachnids.Humans can be treated with the compounds of the invention arid fallwithin the mammal sub-category.

As used herein, the term “CAAX” means a C-terminal peptide sequencewherein C is Cys, A is an amino acid, usually an aliphatic amino acid,and X is another amino acid, usually Leu or Phe.

As used herein, the term “CAAX protein” means a protein comprising aCAAX sequence.

As used herein, the term “XXCC” means a C-terminal peptide sequencewherein C is Cys and X is another amino acid, usually Leu or Phe.

As used herein, the term “XXCC protein” means a protein comprising aXXCC sequence.

As used herein, the term “XCXC” means a C-terminal peptide sequencewherein C is Cys and X is another amino acid, usually Leu or Phe.

As used herein, the term “XCXC protein” means a protein comprising aXCXC sequence.

As used herein, the term “Ras or Ras-like protein” encompasses Rasproteins, brain heterotrimeric G proteins, and other GTP-bindingproteins such as members of the Rho, Rac and Rab family including, butnot limited to, RhoA, RhoB, RhoC, CDC42Hs, Rac1, Rac2, Rap1A and Rap1B.A Ras or Ras-like protein may be a CAAX, XXCC, or XCXC protein. The term“Ras or Ras-like protein” as used herein also encompasses Rheb,inositol-1,4,5,triphosphate-5-phosphatase, and cyclic nucleotidephosphodiesterase and isoforms thereof, including nuclear lamin A and B,fungal mating factors, and several proteins in visual signaltransduction.

As used herein, the term “Ras or Ras-like protein prenylation” means theprenylation of a Ras or Ras-like protein that is catalyzed or caused byGGPTase I, GGPTase II, or FPTase.

As used herein, the term “prenylation inhibitor” means a compound ormixture of compounds that inhibits, restrains, retards, blocks orotherwise affects protein prenylation, preferably Ras or Ras-likeprotein prenylation. A prenylation inhibitor may inhibit, restrain,retard, or otherwise affect the activity of GGPTase I, GGPTase II,and/or FPTase.

As used herein, the term “a pharmaceutically-acceptable salt thereof”refers to salts prepared from pharmaceutically-acceptable nontoxic acidsor bases including inorganic acids and bases and organic acids andbases. Examples of such inorganic acids are hydrochloric, hydrobromic,hydriodic, sulfuric, and phosphoric. Appropriate organic acids may beselected, for example, from aliphatic, aromatic, carboxylic and sulfonicclasses of organic acids, examples of which are formic, acetic,propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic,stearic, sulfanilic, algenic, tartaric, citric and galacturonic.Examples of suitable inorganic bases include metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.Appropriate organic bases may be selected, for example, fromN,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumaine (N-methylglucamine), lysine and procaine.Preferred salts of the compounds of this invention are TFA and acetatesalts.

The phrase “therapeutically effective amount of prenylation inhibitor”as used herein means that amount of prenylation inhibitor which alone orin combination with other drugs provides a therapeutic benefit in thetreatment, management, or prevention of conditions in a plant or animalthat are caused, aggravated or prolonged by Ras or Ras-like proteinprenylation. Such conditions include, but are not limited to, cancer,restenosis, psoriasis, endometriosis, atherosclerosis, ischemia,myocardial ischemic disorders such as myocardial infarction, high serumcholesterol levels, viral infection, fungal infections, yeastinfections, bacteria and protozoa infections, and undesiredangiogenesis, abnormal angiogenesis or abnormal proliferation such as,but not limited to, corneal neovascularization. Other conditions includeabnormal bone resorption and conditions related thereto.

“Alkyl” groups according to the present invention are aliphatichydrocarbons which can be straight, branched or cyclic. Alkyl groupsoptionally can be substituted with one or more substituents, such as ahalogen, alkenyl, alkynyl, aryl, hydroxy, amino, thio, alkoxy, carboxy,oxo or cycloalkyl. There may be optionally inserted along the alkylgroup one or more oxygen, sulfur or substituted or unsubstitutednitrogen atoms. Exemplary alkyl groups include methyl, ethyl, propyl,i-propyl, n-butyl, t-butyl, bicycloheptane (norbornane), cyclobutane,dimethyl-cyclobutane, cyclopentane, cyclohexane, fluoromethyl,difluoromethyl, trifluoromethyl, chloromethyl, trichloromethyl, andpentafluoroethyl. Preferably, alkyl groups have from about 1 to about 20carbon atom chains, more preferably from about 1 to about 10 carbonatoms, still more preferably from about 1 to about 6 carbon atoms, andmost preferably from about 1 to about 4 carbon atoms.

“Aryl” groups are monocyclic or bicyclic carbocyclic or heterocyclicaromatic ring moieties. Aryl groups can be substituted with one or moresubstituents, such as a halogen, alkenyl, alkyl, alkynyl, hydroxy,amino, thio, alkoxy or cycloalkyl.

“Heteroaryl” refers to monocyclic or bicyclic aromatic ring having atleast one heteroatom selected from nitrogen, sulfur, phosphorus andoxygen. Preferred heteroaryls are 5- and 6-membered aromatic rings whichcontain from about 1 to about 3 heteroatoms. Examples of heteroarylgroups include, but are not limited to, pyridinyl, imidazolyl,pyrimidinyl, pyrazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl,tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, thiazolyl,pyrrole, thiophenyl, furanyl, pyridazinyl, isothiazolyl, andS-triazinyl.

“N-heteroaryl” refers to monocyclic or bicyclic aromatic ring having atleast one nitrogen atom in the aromatic ring moiety. ExemplaryN-heteroaryls include, but are not limited to, pyridinyl, imidazolyl,pyrrimidinyl, pyrazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl,tetrazolyl, isoxazolyl, oxazolyl, pyrrolyl, pyrrole, pyridazinyl, andisothiazolyl. Preferably, N-heteroaryl is pyridinyl. More preferably,N-heteroaryl is pyridin-3-yl.

The term “aryl containing at least one nitrogen substituent” refers toan aryl moiety having a substituent such as an amino, including mono-,di-, and tri-alkyl amino groups; amido; or C₁-C₄ alkyl groups having anamino or an amido substituent. Preferably, an “aryl containing at leastone nitrogen substituent” is an aryl moiety having amino, amido or C₁-C₂alkyl having an amino or amido substituent; more preferably amino, amidoor C₁ alkyl having an amino or amido substituent; still more preferablyan amino or amido substituent; and most preferably an amino substituent.

The term “peptoids” or “polypeptoids” refers to poly-(N-substitutedglycine) chains. These peptidomimetic molecules have a number ofparticular advantages as discussed below. For example, peptoids aresynthetic and non-natural polymers with controlled sequences andlengths, that may be made by automated solid-phase organic synthesis toinclude a wide variety of side-chains having different chemicalfunctions. Peptoids have a number of notable structural features incomparison to peptides. For example, peptoids lack amide protons; thus,no intrachain hydrogen-bond network along the polymer backbone ispossible, unless hydrogen-bond donating side-chains are put in thepeptoid chain. In addition, whereas the side-chain (“R”) groups onbiosynthetically produced peptides must be chosen from among the 20amino acids, peptoids can include a wide variety of different,non-natural side-chains because in peptoid synthesis the R group can beintroduced as a primary amine. This is in contrast to synthetic peptidesfor which the incorporation of non-natural side-chains requires the useof non-natural a-protected amino acids. Polypeptoid (or peptoids) can besynthesized in a sequence-specific fashion using an automatedsolid-phase protocol, e.g., the sub-monomer synthetic route. See, forexample, Wallace et al., Adv. Amino Acid Mimetics Peptidomimetics, 1999,2, 1-51 and references cited therein, all of which are incorporatedherein in their entirety by this reference.

The flexibility of sub-monomer synthesis allows attachment ofside-chains that satisfy the requirements of specific needs, e.g.,hydrophilicity or hydrophobicity. Another advantage of the peptoidsynthetic protocol is that it allows easy production of peptoid-peptidechimerae. In a single automated solid-phase protocol, one can alternatethe addition of peptoid monomers with the addition of (α-Fmoc-protectedpeptide monomers, the latter added by standard Fmoc coupling protocolsemploying activating agents such as pyBrop or pyBop (i.e.,1H-benzotriazol-1-yloxy-tris(pyrrolidino)phosphoniumhexafluorophosphate).

Unless otherwise stated, the term “aromatic group” refers to aryl andheteroaryl groups.

The terms “substituted,” “substituted derivative” and “derivative” whenused to describe a chemical moiety means that at least one hydrogenbound to the unsubstituted chemical moiety is replaced with a differentatom or a chemical moiety. Examples of substituents include, but are notlimited to, alkyl, halogen, nitro, cyano, heterocycle, aryl, heteroaryl,amino, amide, hydroxy, ester, ether, carboxylic acid, thiol, thioester,thioether, sulfoxide, sulfone, carbamate, peptidyl, PO₃H₂, and mixturesthereof.

The term “cancer” encompasses, but is not limited to, myeloid leukemia;malignant lymphoma; lymphocytic leukemia; myeloproliferative diseases;solid tumors including benign tumors, adenocarcinomas, and sarcomas; andblood-borne tumors. The term “cancer” as used herein includes, but isnot limited to, cancers of the cervix, breast, bladder, colon, stomach,prostate, larynx, endometrium, ovary, oral cavity, kidney, testis andlung.

The terms “compound of the present invention,” “compound of thisinvention,” “compound of the invention,” “prenylation inhibitor of thepresent invention,” “prenylation inhibitor of this invention,” and“prenylation inhibitor of the invention” are used interchangeably torefer to the compounds and complexes disclosed herein, and to theirpharmaceutically acceptable salts, solvates, hydrates, polymorphs, andclatherates thereof, and to crystalline and non-crystalline formsthereof.

The present invention is based upon the discovery that certainpyrazole-based compounds are potent prenylation inhibitors. Thesecompounds inhibit the activity of one or more of the following: GGPTaseI, GGPTase II, and FPTase. In one particular embodiment, the compoundsof the present invention, under the assay conditions disclosed in theExamples section, have an IC₅₀ value for GGPTase I of about 25 μM orless, more preferably about 10 μM or less, more preferably about 5 μM orless, more preferably about 10 nanomolar or less and most preferablybetween about 1 nanomolar and about 2 nanomolar.

This invention is further based upon the recognition that proteinprenylation, in particular prenylation of CAAX, XXCC and/or XCXCproteins, is associated with a variety of diseases and/or conditions inplants and animals. In animals, such diseases include, but are notlimited to, cancer, restenosis, psoriasis, endometriosis,atherosclerosis, ischemia, myocardial ischemic disorders such asmyocardial infarction, high serum cholesterol levels, viral infection,fungal infections, yeast infections, bacteria and protozoa infections,proliferative disorders, and disorders related to abnormal angiogenesisincluding, but not limited to, corneal neovascularization. In plants,such diseases include yeast and viral infections.

Compounds of the present invention useful for inhibition of proteinprenylation are shown below. It should be recognized that reference to acompound, identification of a general chemical structure or a specificcompound below and in the claims refers to the compound itself, as wellas pharmaceutically acceptable salts thereof. Moreover, to the extentthe structure of elements in the definitions of the R groups permitsmore than one site of attachment to the main structure, preferred sitesof attachment for such elements are shown below in the exemplaryspecific structures an/or are dictated by the various methods ofsynthesis shown below.

One embodiment of the present invention includes compounds that may beused for inhibiting protein prenylation having the general structure ofFormula I:

-   -   Each X is independently C, N, O or S;    -   R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl;    -   R₂ is methyl, pyridine, pyridine-1-oxide, 3-cyanophenyl,        3-aminophenyl, 3-amidinophenyl, 3-dimethylaminophenyl,        2-methylthiazole, 4-methylthiadiazole, thiadiazole,        5-methylisoxazole, pyrazine, pyrimidine, 5-methylimidazole,        5-methylpyrazole, 2-benzylsulfanylpyridine,        6-benzylsulfanylpyridine,CH₂COOH, N(CH₃)₂, CH₂CH₂SCH₃ or        CH₂-piperidinyl;    -   R₃ is absent, H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,        CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,        CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃;    -   R₄ is absent, H, NH₂, CON(CH₃)₂, CO₂H, CN, CH₂OH, CONH₂, CSNH₂,        CONHOH, C(NH)NH₂, CONHNH₂, CONHCH₃, CH₂OCH₃, CONH-cyclohexyl,        CO₂CH₃,

-   -   R₅ is absent, isopropyl, benzyl, 4-trifluoromethylbenzyl,        4-cyanobenzyl, 4-benzoylbenzyl, 3-chlorobenzyl,        pentafluorobenzyl, 3,4-dichlorobenzyl, 2-fluorobenzyl,        4-methoxybenzyl, CH₂CH₂-phenyl, 4-fluorobenzyl, 4-phenylbenzyl,        CH₂-imidazole, CH₂COOH, CH₂CH₂COOH, (CH₂)₄NH₂, CH₂CH₂SCH₃,        4-hydroxybenzyl, CH₂-naphthyl, 4-methylbenzyl, CH₂-indole,        CH₂-thiophene, CH₂-cyclohexane, 4-chlorobenzyl, phenyl,        2-hydroxybenzyl, 4-tertbutoxybenzyl, CH₂-benzylimidazole,        4-aminobenzyl, CH₂-pryid-3-yl, CH₂-pryid-2-yl, CH₂OH,        (CH₂)₃NHC(NH)NH₂ or CH₂CH(CH₃)₂; and,    -   R₆ is H, methyl, ethyl, propyl, isopropyl, CH₂CO₂H, CH₂CO₂Et,        benzyl, or CH₂-(2-methoxynaphthyl); or,    -   R₅ and R₆ together form:

Another embodiment of the present invention includes compounds that maybe used for inhibiting protein prenylation having the general structureof Formula II:

-   -   Each X is independently C, N, O or S;    -   R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl;    -   R₂ is methyl, pyridine, pyridine-1-oxide, 3-cyanophenyl,        3-aminophenyl, 3-amidinophenyl, 3-dimethylaminophenyl,        2-methylthiazole, 4-methylthiadiazole, thiadiazole,        5-methylisoxazole, pyrazine, pyrimidine, 5-methylimidazole,        5-methylpyrazole, 2-benzylsulfanylpyridine,        6-benzylsulfanylpyridine,CH₂COOH, N(CH₃)₂, CH₂CH₂SCH₃ or        CH₂-piperidinyl;    -   R₃ is absent, H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,        CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,        CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃;    -   R₄ is absent, H, NH₂, CON(CH₃)₂, CO₂H, CN, CH₂OH, CONH₂, CSNH₂,        CONHOH, C(NH)NH₂, CONHNH₂, CONHCH₃, CH₂OCH₃, CONH-cyclohexyl,        CO₂CH₃,

-   -   R₅ is absent, isopropyl, benzyl, 4-trifluoromethylbenzyl,        4-cyanobenzyl, 4-benzoylbenzyl, 3-chlorobenzyl,        pentafluorobenzyl, 3,4-dichlorobenzyl, 2-fluorobenzyl,        4-methoxybenzyl, CH₂CH₂-phenyl, 4-fluorobenzyl, 4-phenylbenzyl,        CH₂-imidazole, CH₂COOH, CH₂CH₂COOH, (CH₂)₄NH₂, CH₂CH₂SCH₃,        4-hydroxybenzyl, CH₂-naphthyl, 4-methylbenzyl, CH₂-indole,        CH₂-thiophene, CH₂-cyclohexane, 4-chlorobenzyl, phenyl,        2-hydroxybenzyl, 4-tertbutoxybenzyl, CH₂-benzylimidazole,        4-aminobenzyl, CH₂-pryid-3-yl, CH₂-pryid-2-yl, CH₂OH,        (CH₂)₃NHC(NH)NH₂ or CH₂CH(CH₃)₂; and,    -   R₆ is H, methyl, ethyl, propyl, isopropyl, CH₂CO₂H, CH₂CO₂Et,        benzyl, or CH₂-(2-methoxynaphthyl); or,    -   R₅ and R₆ together form:

Another embodiment of the present invention includes compounds that maybe used for inhibiting protein prenylation having the general structureof Formula III:

-   -   Each X is independently C, N, O or S;    -   R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl;    -   R₂ is methyl, pyridine, pyridine-1-oxide, 3-cyanophenyl,        3-aminophenyl, 3-amidinophenyl, 3-dimethylaminophenyl,        2-methylthiazole, 4-methylthiadiazole, thiadiazole,        5-methylisoxazole, pyrazine, pyrimidine, 5-methylimidazole,        5-methylpyrazole, 2-benzylsulfanylpyridine,        6-benzylsulfanylpyridine,CH₂COOH, N(CH₃)₂, CH₂CH₂SCH₃ or        CH₂-piperidinyl;    -   R₃ is absent, H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,        CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,        CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃;    -   R₄ is absent, H, NH₂, CON(CH₃)₂, CO₂H, CN, CH₂OH, CONH₂, CSNH₂,        CONHOH, C(NH)NH₂, CONHNH₂, CONHCH₃, CH₂OCH₃, CONH-cyclohexyl,        CO₂CH₃,

-   -   R₅ is absent, isopropyl, benzyl, 4-trifluoromethylbenzyl,        4-cyanobenzyl, 4-benzoylbenzyl, 3-chlorobenzyl,        pentafluorobenzyl, 3,4-dichlorobenzyl, 2-fluorobenzyl,        4-methoxybenzyl, CH₂CH₂-phenyl, 4-fluorobenzyl, 4-phenylbenzyl,        CH₂-imidazole, CH₂COOH, CH₂CH₂COOH, (CH₂)₄NH₂, CH₂CH₂SCH₃,        4-hydroxybenzyl, CH₂-naphthyl, 4-methylbenzyl, CH₂-indole,        CH₂-thiophene, CH₂-cyclohexane, 4-chlorobenzyl, phenyl,        2-hydroxybenzyl, 4-tertbutoxybenzyl, CH₂-benzylimidazole,        4-aminobenzyl, CH₂-pryid-3-yl, CH₂-pryid-2-yl, CH₂OH,        (CH₂)₃NHC(NH)NH₂ or CH₂CH(CH₃)₂; and,    -   R₆ is H, methyl, ethyl, propyl, isopropyl, CH₂CO₂H, CH₂CO₂Et,        benzyl, or CH₂-(2-methoxynaphthyl); or,    -   R₅ and R₆ together form:

Another embodiment of the present invention includes compounds that maybe used for inhibiting protein prenylation having the general structureof Formula IV:

-   -   Each X is independently C, N, O or S;    -   R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl;    -   R₂ is methyl, pyridine, pyridine-1-oxide, 3-cyanophenyl,        3-aminophenyl, 3-amidinophenyl, 3-dimethylaminophenyl,        2-methylthiazole, 4-methylthiadiazole, thiadiazole,        5-methylisoxazole, pyrazine, pyrimidine, 5-methylimidazole,        5-methylpyrazole, 2-benzylsulfanylpyridine,        6-benzylsulfanylpyridine,CH₂COOH, N(CH₃)₂, CH₂CH₂SCH₃ or        CH₂-piperidinyl;    -   R₃ is absent, H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,        CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,        CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃;    -   R₄ is absent, H, NH₂, CON(CH₃)₂, CO₂H, CN, CH₂OH, CONH₂, CSNH₂,        CONHOH, C(NH)NH₂, CONHNH₂, CONHCH₃, CH₂OCH₃, CONH-cyclohexyl,        CO₂CH₃,

-   -   R₅ is absent, isopropyl, benzyl, 4-trifluoromethylbenzyl,        4-cyanobenzyl, 4-benzoylbenzyl, 3-chlorobenzyl,        pentafluorobenzyl, 3,4-dichlorobenzyl, 2-fluorobenzyl,        4-methoxybenzyl, CH₂CH₂-phenyl, 4-fluorobenzyl, 4-phenylbenzyl,        CH₂-imidazole, CH₂COOH, CH₂CH₂COOH, (CH₂)₄NH₂, CH₂CH₂SCH₃,        4-hydroxybenzyl, CH₂-naphthyl, 4-methylbenzyl, CH₂-indole,        CH₂-thiophene, CH₂-cyclohexane, 4-chlorobenzyl, phenyl,        2-hydroxybenzyl, 4-tertbutoxybenzyl, CH₂-benzylimidazole,        4-aminobenzyl, CH₂-pryid-3-yl, CH₂-pryid-2-yl, CH₂OH,        (CH₂)₃NHC(NH)NH₂ or CH₂CH(CH₃)₂;    -   R₆ is H, methyl, ethyl, propyl, isopropyl, CH₂CO₂H, CH₂CO₂Et,        benzyl, or CH₂-(2-methoxynaphthyl); or,    -   R₅ arid R₆ together form:

-   -   R₇ is absent, NH(CH₂)₃, NHCOCH₂, NHCOCH₂CH₂, NH-piperidine-CH₂,        NHCO-pyridine, NHCONH-cyclohexane, O—CH₂, O—(CH₂)₄,        CONH-cyclopentane, CH₂NHCH₂CH₂, CH₂N⁺H₂Cl⁻CH₂CH₂,        CH₂N(CH₂COOH)CH₂CH₂, CH₂N(CH₂CH₂N(CH₃)₂)CH₂CH₂, NHCH(CH₃)CH₂CH₂,        CH₂-Piperidine, CH₂NHCH₂CH(CH₃)CH₂, CH₂NHCH(CONH₂),        CH₂N(CH₃)CH₂CH₂, CH₂N(CH₂CH₃)CH₂CH₂,        CH₂N(CH₂CH₂-morpholine)CH₂CH₂, OCH₂CH(OH)CH₂, OCH₂COCH₂,        OCH₂CH₂CH(OH), OCH₂CH₂C(CH₃)₂, CH₂N(CH₂CH₂OCH₃)CH₂CH₂,

Another embodiment of the present invention includes compounds that maybe used for inhibiting protein prenylation having the general structureof Formula V:

-   -   Each X is independently C, N, O or S;    -   R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl;    -   R₂ is NH₂, CO₂CH₃, C(O)N₃, C(O)NH-cyclohexane, 4-aminophenyl,        CH₂CNHNH₂, O(CH₂)₃CONHCOCH(NH₂)benzyl, OH, OCH₂CH₃, (CH₂)₄COOH,        O(CH₂)₃COOH, O(CH₂)₄COOH, O(CH₂)₃COOCH₂CH₃, CH₂NHCH₂CH₂N(CH₃)₂,        benzamide, benzoic acid, thiophene carboxylic acid or        urea-cyclopropane carboxylic acid; and,    -   R₃ is absent, H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,        CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,        CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃

Another embodiment of the present invention includes compounds that maybe used for inhibiting protein prenylation having the general structureof Formula VI:

-   -   Each X is independently C or N;    -   R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl;    -   R₂ is methyl, pyridine, pyridine-1-oxide, 3-cyanophenyl,        3-aminophenyl, 3-amidinophenyl, 3-dimethylaminophenyl,        2-methylthiazole, 4-methylthiadiazole, thiadiazole,        5-methylisoxazole, pyrazine, pyrimidine, 5-methylimidazole,        5-methylpyrazole, 2-benzylsulfanylpyridine,        6-benzylsulfanylpyridine,CH₂COOH, N(CH₃)₂, CH₂CH₂SCH₃ or        CH₂-piperidinyl;    -   R₃ is absent, H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,        CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,        CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃;    -   R₄ is absent, H, NH₂, CON(CH₃)₂, CO₂H, CN, CH₂OH, CONH₂, CSNH₂,        CONHOH, C(NH)NH₂, CONHNH₂, CONHCH₃, CH₂OCH₃, CONH-cyclohexyl,        CO₂CH₃,

-   -   R₅ is absent, isopropyl, benzyl, 4-trifluoromethylbenzyl,        4-cyanobenzyl, 4-benzoylbenzyl, 3-chlorobenzyl,        pentafluorobenzyl, 3,4-dichlorobenzyl, 2-fluorobenzyl,        4-methoxybenzyl, CH₂CH₂-phenyl, 4-fluorobenzyl, 4-phenylbenzyl,        CH₂-imidazole, CH₂COOH, CH₂CH₂COOH, (CH₂)₄NH₂, CH₂CH₂SCH₃,        4-hydroxybenzyl, CH₂-naphthyl, 4-methylbenzyl, CH₂-indole,        CH₂-thiophene, CH₂-cyclohexane, 4-chlorobenzyl, phenyl,        2-hydroxybenzyl, 4-tertbutoxybenzyl, CH₂-benzylimidazole,        4-aminobenzyl, CH₂-pryid-3-yl, CH₂-pryid-2-yl, CH₂OH,        (CH₂)₃NHC(NH)NH₂ or CH₂CH(CH₃)₂; and,    -   R₆ is H, methyl, ethyl, propyl, isopropyl, CH₂CO₂H, CH₂CO₂Et,        benzyl, or CH₂-(2-methoxynaphthyl); or,    -   R₅ and R₆ together form:

Another embodiment of the present invention includes compounds that maybe used for inhibiting protein prenylation having the general structureof a formula selected from the group consisting of Formulae VII-X:

-   -   wherein R₁ is phenyl, benzyl, methyl, ethyl, propyl, pyrimidine,        3,4-dimethylphenyl, 3-chloropyridazine, 2,4-dimethylpyrimidine,        3,4-difluorophenyl, 3,4-dichlorophenyl, 3,5-dichlorophenyl,        CH₂CF₃, 4-trifluoromethylphenyl, 4-nitrophenyl, 4-bromophenyl,        3-bromophenyl, 4-methylphenyl, 4-methoxyphenyl,        4-chloro-2-methylphenyl, 4-fluorophenyl, 4-sulfonamidophenyl,        3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,        3,5-difluorophenyl, 4-aminophenyl, 1,3-dimethyl pyrazole,        ethanol, or 3,4-methylenedioxyphenyl.

Specific compounds of the present invention useful for proteinprenylation are shown below in Table 1.

TABLE 1 1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

1040

1041

1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

1080

1081

1082

1083

1084

1085

1086

1087

1088

1089

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

1100

1101

1102

1103

1104

1105

1106

1107

1108

1109

1110

1111

1112

1113

1114

1115

1116

1117

1118

1119

1120

1121

1122

1123

1124

1125

1126

1127

1128

1129

1130

1131

1132

1133

1134

1135

1136

1137

1138

1139

1140

1141

1142

1143

1144

1145

1146

1147

1148

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158

1159

1160

1161

1162

1163

1164

1165

1166

1167

1168

1169

1170

1171

1172

1173

1174

1175

1176

1177

1178

1179

1180

1181

1182

1183

1184

1185

1186

1187

1188

1189

1190

1191

1192

1193

1194

1195

1196

1197

1198

1199

1200

1201

1202

1203

1204

1205

1206

1207

1208

1209

1210

1211

1212

1213

1214

1215

1216

1217

1218

1219

1220

1221

1222

1223

1224

1225

1226

1227

1228

1229

1230

1231

1232

1233

1234

1235

1236

1237

1238

1239

1240

1241

1242

1243

1244

1245

1246

1247

1248

1249

1250

1251

1252

1253

1254

1255

1256

1257

1258

1259

1260

1261

1262

1263

1264

1265

1266

1267

1268

1269

1270

1271

1272

1273

1274

1275

1276

The compounds of the present invention can be synthesized from readilyavailable starting materials. Various substituents on the compounds ofthe present invention can be present in the starting compounds, added toany one of the intermediates or added after formation of the finalproducts by known methods of substitution or conversion reactions. Ifthe substituents themselves are reactive, then the substituents canthemselves be protected according to the techniques known in the art. Avariety of protecting groups are known in the art, and can be employed.Examples of many of the possible groups can be found in ProtectiveGroups in Organic Synthesis, 2nd edition, T. H. Greene and P. G. M.Wuts, John Wiley & Sons, New York, N.Y., 1991, which is incorporatedherein in its entirety by this reference. For example, nitro groups canbe added by nitration and the nitro group can be converted to othergroups, such as amino by reduction, and halogen by diazotization of theamino group and replacement of the diazo group with halogen. Acyl groupscan be added by Friedel-Crafts acylation. The acyl groups can then betransformed to the corresponding alkyl groups by various methods,including the Wolff-Kishner reduction and Clemmenson reduction. Aminogroups can be alkylated to form mono- and di-alkylamino groups; andmercapto and hydroxy groups can be alkylated to form correspondingethers. Primary alcohols can be oxidized by oxidizing agents known inthe art to form carboxylic acids or aldehydes, and secondary alcoholscan be oxidized to form ketones. Thus, substitution or alterationreactions can be employed to provide a variety of substituentsthroughout the molecule of the starting material, intermediates, or thefinal product, including isolated products.

Since the compounds of the present invention can have certainsubstituents that are necessarily present, the introduction of eachsubstituent is, of course, dependent on the specific substituentsinvolved and the chemistry necessary for their formation. Thus,consideration of how one substituent would be affected by a chemicalreaction when forming a second substituent would involve techniquesfamiliar to one of ordinary skill in the art. This would further bedependent on the ring involved.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. It is to be understood that thepresent invention encompasses any racemic, optically-active,regioisomeric or stereoisomeric form, or mixtures thereof, of a compoundof the invention, which possess the useful properties described herein,it being well known in the art how to prepare optically active forms(for example, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine prenylation inhibitor activityusing the standard tests described herein, or using other similar testswhich are well known in the art. It is also to be understood that thescope of this invention encompasses not only the various isomers whichmay exist but also the various mixtures of isomers which may be formed.For example, if the compound of the present invention contains one ormore chiral centers, the compound can be synthesized enantioselectivelyor a mixture of enantiomers and/or diastereomers can be prepared andseparated. The resolution of the compounds of the present invention,their starting materials and/or the intermediates may be carried out byknown procedures, e.g., as described in the four volume compendiumOptical Resolution Procedures for Chemical Compounds: Optical ResolutionInformation Center, Manhattan College, Riverdale, N.Y., and inEnantiomers, Racemates and Resolutions, Jean Jacques, Andre Collet andSamuel H. Wilen; John Wiley & Sons, Inc., New York, 1981, which isincorporated in its entirety by this reference. Basically, theresolution of the compounds is based on the differences in the physicalproperties of diastereomers by attachment, either chemically orenzymatically, of an enantiomerically pure moiety resulting in formsthat are separable by fractional crystallization, distillation orchromatography.

When the compound of the present invention contains an olefin moiety andsuch olefin moiety can be either cis- or trans-configuration, thecompound can be synthesized to produce cis- or trans-olefin,selectively, as the predominant product. Alternatively, the compoundcontaining an olefin moiety can be produced as a mixture of cis- andtrans-olefins and separated using known procedures, for example, bychromatography as described in W. K. Chan, et al., J. Am. Chem. Soc.,1974, 96, 3642, which is incorporated herein in its entirety by thisreference.

The compounds of the present invention form salts with acids when abasic amino function is present and salts with bases when an acidfunction, e.g., carboxylic acid or phosphonic acid, is present. All suchsalts are useful in the isolation and/or purification of the newproducts. Of particular value are the pharmaceutically acceptable saltswith both acids and bases. Suitable acids and bases are described above.In addition, hydrated, solvated and/or anhydrous forms of compoundsdisclosed herein are also encompassed in the present invention.

The compounds of present invention may be prepared by both conventionaland solid phase synthetic techniques known to those skilled in the art.Useful conventional techniques include those disclosed by U.S. Pat. Nos.5,569,769 and 5,242,940, and PCT publication No. WO 96/37476, each ofwhich are incorporated herein in their entirety by this reference.

Combinatorial synthetic techniques, however, are particularly useful forthe synthesis of the compounds of the present invention. See, e.g.,Brown, Contemporary Organic Synthesis, 1997, 216; Felder and Poppinger,Adv. Drug Res., 1997, 30, 111; Balkenhohl et al., Angew. Chem. Int. Ed.Engl., 1996, 35, 2288; Hermkens et al., Tetrahedron, 1996, 52, 4527;Hermkens et al., Tetrahedron, 1997, 53, 5643; Thompson et al., Chem.Rev., 1996, 96, 555; and Nefzi et al., Chem. Rev., 1997, 2, 449-472.

One solid phase synthetic approach useful for preparing compounds ofthis invention is described by Marzinzik and Felder, Tetrahedron Lett.,1996, 37, 1003-1006, and Marzinzik and Felder, J. Org. Chem., 1998, 63,723-727. A general adaptation of this approach is shown in the syntheticscheme of FIG. 1. Referring to FIG. 1, <A>, <B>, <C> and <D> representreaction conditions suitable for the formation of the desired productsor intermediates represented by Formulas (a)-(d); W, X, Y and Zconstitute moieties within the compounds of the present invention asdefined above, and R is a halogenated phenyl.

As shown in FIG. 1, an appropriate compound is attached to a resin orother solid support under reaction conditions <A> to form a complex ofFormula (a). Appropriate reaction conditions and solid supports are wellknown to those skilled in the art. The immobilized compound of Formula(a) is then combined with a suitable reactant comprising the moieties Rand Z to yield a compound of Formula (b). Suitable reactants for theformation of the compound of Formula (b) include, for example, ketoacids and the like, and depend upon the nature of the leaving group Land reaction conditions <B>. Suitable reactants and reaction conditionsare well known to those skilled in the art. See, e.g., March, AdvancedOrganic Chemistry 3^(rd) ed., John Wiley & Sons, Inc., New York, N.Y.,1985, pp. 435-437, which is incorporated herein by reference.

According to FIG. 1, the immobilized compound of Formula (b) is thensubjected to reaction conditions <C> to form the pyrazole compound ofFormula (c), wherein R is typically as defined above, or a precursorthereto. Reaction conditions <C> are also well known to one of ordinaryskill in the art. See, e.g., March, Advanced Organic Chemistry 3^(rd)ed., John Wiley & Sons, Inc., New York, N.Y., 1985, p. 804, which isincorporated herein by this reference.

Finally, the compound of Formula (c) is cleaved from the resin underreaction conditions <D> that are well known to those skilled in the artto yield the final product of Formula (d) which, if desired, may undergopurification, crystallization or recrystallization, or further reactionsto form compounds of this invention.

A particular embodiment of this approach is presented in the synthesisscheme shown in FIG. 2 where AA is a natural or synthetic amino acid,and X, Y, Z and R are those defined above.

In the first step of the scheme of FIG. 2, the protected amine groupsbound to the resin are deprotected and reacted with a protected naturalor synthetic amino acid under suitable conditions. Although both theresin-bound amine and the amino acid moiety in FIG. 2 are protected withFmoc, other protecting groups well known to those skilled in the art mayalso be used. See, for example, Protective Groups in Organic Synthesis,2nd edition, T. H. Greene and P. G. M. Wuts, John Wiley & Sons, NewYork, N.Y., 1991, which is incorporated in its entirety by thisreference.

Removal of the amino acid protecting group and reacting the resultingfree amine with a keto acid affords the methyl ketone compound shown inFIG. 2. The third step of FIG. 2 can be carried out using any of themethods known to those of ordinary skill in the art of organicchemistry, including a Claisen condensation reaction. The conditionsmost suitable for this reaction may be determined using compounds suchas ethyl benzoate, such optimization may be necessary in some cases toensure that the reaction occurs without appreciable formation of sideproducts. This reaction is preferably done using dimethylacetamide (DMA)as a solvent.

The fourth step involves formation of the pyrazole ring moiety, forexample, by reacting the 1,3-diketone with an appropriately substitutedhydrazine. The final products may be cleaved from the solid-support byconventional means.

FIGS. 3, 4 and 5 show synthetic schemes for the production of ether,aromatic and amine groups, respectively, that link the aromatic ringhaving two or three heteroatoms to an amide group that may be furthersubstituted to form prenylation inhibitor compounds of the presentinvention.

FIG. 6 shows a synthetic scheme for making substitutions at the4-position of a 5-membered aromatic ring having two or three heteroatomswithin prenylation inhibitor structures of the present invention.

Whether or not formed using the approaches shown in FIGS. 1-6, thecompounds of the present invention that are basic in nature are capableof forming a wide variety of different salts with various inorganic andorganic acids. Although such salts must be pharmaceutically acceptablein order to be administered to organisms, it may be desirable toinitially isolate compounds of the present invention from reactionmixtures as pharmaceutically unacceptable salts, which are thenconverted back to the free base compounds by treatment with an alkalinereagent, and subsequently converted to pharmaceutically acceptable acidaddition salts. The acid addition salts of the basic compounds of thisinvention are readily prepared by treating the compounds withsubstantially equivalent amounts of chosen mineral or organic acids inaqueous solvent mediums, or in suitable organic solvents such asmethanol and ethanol. Upon careful evaporation of these solvents, thedesired solid salts are readily obtained. Desired salts can also beprecipitated from solutions of the free base compounds in organicsolvents by adding to the solutions appropriate mineral or organicacids.

Those compounds of the present invention that are acidic in nature aresimilarly capable of forming base salts with various cations. As above,when a pharmaceutically acceptable salt is required, it may be desirableto initially isolate a compound of the present invention from a reactionmixture as a pharmaceutically unacceptable salt, which can then beconverted to a pharmaceutically acceptable salt in a process analogousto that described above. Examples of base salts include alkali metal oralkaline-earth metal salts and particularly sodium, amine and potassiumsalts. These salts are all prepared by conventional techniques. Thechemical bases used to prepare the pharmaceutically acceptable basesalts of this invention are those which form non-toxic base salts withthe acidic compounds of the present invention. Such nontoxic base saltsinclude those derived from pharmacologically acceptable cations such assodium, potassium, calcium, magnesium, and various amine cations. Thesesalts can easily be prepared by treating the corresponding acidiccompounds with an aqueous solution containing the desiredpharmacologically acceptable bases and then evaporating the resultingsolution to dryness, preferably under reduced pressure. They may also beprepared by mixing lower alkanolic solutions to dryness in the samemanner as before. In either case, stoichiometric quantities of reagentsare preferably employed in order to ensure completeness of reaction andmaximum yields of the desired final product.

This invention encompasses both crystalline and non-crystalline (e.g.,amorphous) forms of the salts of the compounds of this invention. Thesesalts can be used to increase the solubility or stability of thecompounds disclosed herein. They may also aid in the isolation andpurification of the compounds.

Suitable methods of synthesizing the compound of the present inventionmay yield mixtures of regioisomers and/or diastereomers. These mixtures,which are encompassed by the compounds and methods of the presentinvention, can be separated by any means known to those skilled in theart. Suitable techniques include high performance liquid chromatography(HPLC) and the formation and crystallization of chiral salts. See, e.g.,Jacques et al., Enantiomers, Racemates and Resolutions,Wiley-Interscience, New York, N.Y., 1981; Wilen et al., Tetrahedron,1977, 33, 2725; Eliel, Stereochemistry of Carbon Compounds, McGraw-Hill,New York, N.Y., 1962; and Wilen, Tables of Resolving Agents and OpticalResolutions, Eliel, ed., Univ. of Notre Dame Press, Notre Dame, Ind.,1972, p. 268. The resulting enantiomerically enriched compounds areencompassed by the present invention.

The ability of the compounds of the present invention to inhibit proteinprenylation of, for example, Ras or Ras-like proteins, may be determinedby methods known to those skilled in the art such as the methods shownin the Examples below, and by methods disclosed in the referencesincorporated herein. In certain embodiments of the present invention,compounds of the present invention are inhibitory in the GGPTase I assaydescribed in detail in Example 4. For example, compounds of the presentinvention, at a concentration of 10 μM in the GGPTase I assay describedin Example 4, preferably show a percent inhibition of at least about20%, more preferably at least about 35% and more preferably at leastabout 50%. GGPTase I may be prepared and purified according to themethod described by Zhang et al., J. Biol. Chem., 1994, 9, 23465-23470,and U.S. Pat. No. 5,789,558, which is incorporated herein in itsentirety by this reference. GGPTase II may be prepared by a method asdisclosed in, for example, Johannes et al., Eur. J. Biochem., 1996, 239,362-368; and Witter and Poulter, Biochemistry, 1996, 35, 10454-10463,all of which are incorporated herein in their entirety by thisreference. FPTase may be prepared and purified by methods such as thosedisclosed by U.S. Pat. Nos. 5,141,851 and 5,578,477, both of which areincorporated herein in their entirety by this reference.

The compounds of the present invention can be used for inhibitingprotein prenylation by contacting an isoprenoid transferase with thecompound. The compound can be contacted with a cell, in vitro or exvivo, and be taken up by the cell. The compounds of the presentinvention can also be administered to an organism to achieve a desiredeffect. An organism may be a plant or an animal, preferably a mammal,and more preferably a human.

For inhibiting protein prenylation in an animal, the compound of thepresent invention can be administered in a variety of forms adapted tothe chosen route of administration, i.e., orally or parenterally.Parenteral administration in this respect includes administration by thefollowing routes: intravenous; intramuscular; subcutaneous; intraocular;intrasynovial; transepithelially including transdermal, ophthalmic,sublingual and buccal; topically including ophthalmic, dermal, ocular,rectal and nasal inhalation via insufflation and aerosol;intraperitoneal; and rectal systemic.

In one particular embodiment of the present invention, proteinprenylation inhibition is used to treat or prevent conditions in anorganism due to Ras or Ras-like protein prenylation. In animals, suchdiseases include, but are not limited to, cancer, restenosis, psoriasis,endometriosis, proliferative disorders, atherosclerosis, ischemia,myocardial ischemic disorders such as myocardial infarction, high serumcholesterol levels, viral infection, fungal infections, yeast infectionsor corneal neovascularization. In plants, such diseases include yeastand viral infections.

The method of the present invention can also include the administrationof a dosage form comprising at least one compound of the presentinvention alone or in combination with other drugs or compounds. Otherdrugs or compounds that may be administered in combination with thecompounds of the present invention may aid in the treatment of thedisease or disorder being treated, or may reduce or mitigate unwantedside-effects that may result from the administration of the compounds.

The magnitude of a prophylactic or therapeutic dose of a compound of thepresent invention used in the prevention, treatment, or management of adisorder or condition can be readily determined by one of skill in theart using in vitro and in vivo assays such as those described below. Asthose of skill in the art will readily recognize, however, the magnitudeof a prophylactic or therapeutic dose of a prenylation inhibitor willvary with the severity of the disorder or condition to be treated, theroute of administration, and the specific compound used. The dose, andperhaps the dose frequency, will also vary according to the age, bodyweight, and response of the individual patient.

Typically, the physician will determine the dosage of the presenttherapeutic agents which will be most suitable for prophylaxis ortreatment and it will vary with the form of administration and theparticular compound chosen, and also, it will vary with the particularpatient under treatment. The physician will generally wish to initiatetreatment with small dosages by small increments until the optimumeffect under the circumstances is reached. The therapeutic dosage cangenerally be from about 0.1 to about 1000 mg/day, and preferably fromabout 10 to about 100 mg/day, or from about 0.1 to about 50 mg/Kg ofbody weight per day and preferably from about 0.1 to about 20 mg/Kg ofbody weight per day and can be administered in several different dosageunits. Higher dosages, on the order of about 2× to about 4×, may berequired for oral administration. In another aspect, the therapeuticdosage can be sufficient to achieve blood levels of the therapeuticagent of between about 5 micromolar and about 10 micromolar.

In one exemplary application, a suitable amount of a compound of thepresent invention is administered to a mammal undergoing treatment forcancer. Administration occurs in an amount of between about 0.1 mg/kgbody weight to about 20 mg/kg body weight per day, preferably betweenabout 0.5 mg/kg body weight to about 10 mg/kg body weight per day.

In another exemplary application, a suitable amount of a compound ofthis invention is administered to a mammal undergoing treatment foratherosclerosis. The magnitude of a prophylactic or therapeutic dose ofthe compound will vary with the nature and severity of the condition tobe treated, and with the particular compound and its route ofadministration. In general, however, administration of a compound of thepresent invention for treatment of atherosclerosis occurs in an amountof between about 0.1 mg/kg body weight to about 100 mg/kg of body weightper day, preferably between about 0.5 mg/kg body weight to about 10mg/kg of body weight per day.

It is recommended that children and patients aged over 65 yearsinitially receive low doses, and that they then be titrated based onindividual response(s) or blood level(s). It may be necessary to usedosages outside the ranges identified above in some cases as will beapparent to those skilled in the art. Further, it is noted that theclinician or treating physician will know how and when to adjust,interrupt, or terminate therapy in conjunction with individual patientresponse.

When used to inhibit protein prenylation in plants, the compounds of thepresent invention may be administered as aerosols using conventionalspraying techniques, or may be mixed or dissolved in the food, soiland/or water provided to the plants. Other methods of administrationknown in the art are also encompassed by the invention.

The active compound can be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it can beenclosed in hard or soft shell gelatin capsules, or it can be compressedinto tablets, or it can be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipient and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparation can contain at leastabout 0.1% of active compound. The percentage of the compositions andpreparation can, of course, be varied and can conveniently be betweenabout 1% to about 10% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared such that an oral dosageunit form contains from about 1 to about 1000 mg of active compound.

The tablets, troches, pills, capsules and the like can also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin can be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it can contain, in addition to materials of theabove type, a liquid carrier. Various other materials can be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules can be coated with shellac,sugar or both. A syrup or elixir can contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed.

The active compound can also be administered parenterally. Solutions ofthe active compound as a free base or pharmacologically acceptable saltcan be prepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersion can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluidsuch that it is possible to be delivered by syringe. It can be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent of dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g., sugars or sodium chloride. Prolonged absorption of theinjectable compositions may be accomplished by the inclusion of agentsdelaying absorption in the injectable preparation, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredient into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying technique whichyield a powder of the active ingredient plus any additional desiredingredient from previously sterile-filtered solution thereof.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are employed. If desired, tablets may be coatedby standard aqueous or nonaqueous techniques.

In addition to the common dosage forms set out above, the compounds ofthe present invention may also be administered by controlled releasemeans and/or delivery devices capable of releasing the active ingredient(prenylation inhibitor) at the required rate to maintain constantpharmacological activity for a desirable period of time. Such dosageforms provide a supply of a drug to the body during a predeterminedperiod of time and thus maintain drug levels in the therapeutic rangefor longer periods of time than conventional non-controlledformulations. Examples of controlled release pharmaceutical compositionsand delivery devices that may be adapted for the administration of theactive ingredients of the present invention are described in U.S. Pat.Nos.: 3,847,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200; 4,008,719;4,687,610; 4,769,027; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,566; and 5,733,566, the disclosures of whichare incorporated herein in their entirety by this reference.

Pharmaceutical compositions for use in the methods of the presentinvention may be prepared by any methods known in the pharmaceuticalsciences. Such methods are well known to the art and as described, forexample, in Remington: The Science and Practice of Pharmacy, Lippincott,Williams & Wilkins, pubs, 20th edition (2000). All of these methodsinclude the step of bringing the active ingredient into association withthe carrier that constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product intothe desired presentation.

For example, a tablet may be prepared by compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as powder or granules, optionallymixed with a binder, lubricant, inert diluent, surface active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1

This example illustrates methods for synthesizing compounds of thepresent invention.

Coupling to Polystyrene Rink Resin

About 42 grams (g) of Fmoc-protected Rink polystyrene resin and about100 milliliter (ml) of dimethylformamide (DMF) were combined in a 500 mlpeptide vessel and shaken for about 5 minutes. The DMF was removed,about 200 ml of 20% piperidine in DMF was added to the vessel, and themixture was shaken for 30 minutes. This step was repeated prior to thesolvent being removed. Following removal of the solvent, the resin wasdeprotected by being washed 3 times with 30 ml of DMF and twice with 200ml of 1-methyl-2-pyrrolidinone (NMP). The resin was then dried for about1 hour in vacuo. About 2 equivalents of an amino acid, about 2equivalents of benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP), about 2 equivalents ofN-hydroxybenzotriazole (HOBt), and about 200 ml of NMP were mixed in a250 ml beaker. Before addition to the peptide vessel, containing thedeprotected Rink resin, about 4 equivalents of diisopropylethylamine(DIEA) was added to the mixture and stirred for about 1 minute. Themixture was then shaken for about 2 hours in the peptide vessel. Afterthis time, the solvent was removed and the resin was washed 3 times withabout 200 ml of NMP and 3 times with about 200 ml of dichloromethane(DCM). A ninhydrin test was performed, using standard methods, todetermine if amide formation was complete. Once the coupling wascomplete, the resin was dried overnight in vacuo.

About 5 g of the resin was suspended in about 30 ml of DMF, placed in a50 ml syringe with a polyethylene filter (available from POREXTechnologies, Fairburn, Ga.) and shaken for about 5 minutes. The DMF wasremoved, about 30 ml of 20% piperidine in DMF was added to the syringe,and the mixture was shaken for another 30 minutes. This step wasrepeated before the solvent was removed and the deprotected resin waswashed 3 times with about 30 ml of DMF and 2 times with about 30 ml ofDCM. About 1.5 equivalents of ketoacid, about 1.8 equivalents of PyBOP,about 1.8 equivalents of HOBt and about 30 ml of NMP (30 ml) werecombined. About 4 equivalents of DIEA was added to the mixture andstirred for about 1 minute. The mixture was added to the syringe andshaken for about 16 hours. After this time the solvent was removed andthe resin was washed 3 times with about 30 ml of DMF and 3 times withabout 30 ml of DCM. A ninhydrin test was used to determine if amideformation was complete. Once the coupling was complete, the resin wasdried overnight in vacuo.

About 2 g of resin complex from above was placed in a 35 ml thick-walledglass ACE pressure tube with about 10 equivalents of methyl nicotinateand about 25 ml of dimethylacetamide (DMA) and then vortexed for about 1minute. About 30 equivalents of 60% NaH in oil was added over an about30 minute period under controlled conditions; continuous vortexing, N₂blanket, periodic capping and venting. The mixture was very exothermic.The pressure tube was sealed and rotated from about 85° C. to about 90°C. for about 1 hour. The tube was allowed to cool to about 25° C. in theincubator, chilled to about 0° C., and opened behind a Plexiglas shield.The resin, with residual NaH, was slowly poured over about a 10 minuteperiod into a 500 ml peptide vessel containing about 50 ml of 15% HOAc(aq). The remaining NaH was quenched. Following the quenching, the resinwas washed with about 50 ml of 15% HOAc (aq), then 2 times with about 50ml of DMF, 2 times with about 50 ml of EtOAc, 1 time with about 50 ml ofisopropanol, 1 time with about 50 ml of MeOH, and then dried overnightin vacuo.

Library Production

About 0.05 g of each resin-bound complex set from above was dispensedinto discrete wells of a 96-well polypropylene plate (PolyfiltronicsUnifilter; 0.8 ml volume; 10 μm polypropylene filter) using a repeaterpipette and a 1:1 DMF:chloroform colloid solution of the resin (yielding48×0.25 ml aliquots). The resin was then washed 2 times with about 0.5ml of dichloromethylene and dried using a 96-well plate vacuum box.

The bottom of the 96-well plate was sealed with a TiterTop and securedto the bottom of a 96-well plate press apparatus. An about 0.7 Msolution of a selected hydrazine or substituted hydrazine in about 0.6ml of 2:1:1 DMF:mesitylene:MeOH was added to individual wells in theplate using a BioHit 8-channel pipetter. The top of the 96-well platewas sealed with another TiterTop, and the 96-well press apparatus wassealed. The plate apparatus was rotated overnight at 25° C. Followingremoval of the plate from the apparatus, the solvent was drained and theresin was washed 4 times with about 0.4 ml DMF, 2 times with about 0.4ml MeOH, 3 times with about 0.4 ml methylene chloride, and then driedfor about 1 hour using the 96-well vacuum box.

Cleavage of Product from Polystyrene Rink Resin:

About 0.4 ml of 1:1 trifluoroacetic acid (TFA):methylene chloride wasadded to each well of a semi-sealed 96-well in the 96-well plateapparatus. The 96-well plate was then shaken at 300 rpm for about 30minutes. Using the 96-well plate vacuum box, the solvent was transferredto a marked Beckman 96-well plate. The cleavage process was repeatedtwice with 1:1 TFA:DCM and the resin was washed with 1:1 acetone:DCM.The solvent in the Beckman 96-well plate was evaporated and theremaining product lyophilized 3 times with 1:1 acetonitrile:water.

¹H and ¹³C NMR spectra were obtained on a Bruker AM-250 at 250 MHz and62.9 MHz, respectively, using DMSO-d₆ as the solvent. All peaks werereferenced to the DMSO quintet at 2.49 ppm.

Molecular weight determinations were made using a PE-Sciex API 100 MSbased detector (available from Sciex, Concord, Ontario) equipped with anIon Spray Source. Flow Injection Analysis was carried out using aHTS-PAL auto sampler (available from CTC Analytics, Zwingen,Switzerland) and a HP 1100 binary pump (available from Hewlett-Packard,Palo Alto, Calif.).

The analyte was diluted to about 0.25 ml with 1:1 MeOH/CH₃CN containing1% HOAc. About 25 μL of the analyte sample was directly infused into theIon Source at about 70 μL/1 minutes. Electron spray ionization (ESI)mass spectra was acquired in the positive ion mode. The ion-spray needlewas kept at about 4500 V and the orifice and ring potentials were atabout 50 V and about 300V, respectively. The mass range of 150-650 Dawas scanned using a step size of 0.1 Da and a dwell time of 0.6 msresulting in a total scan time of about 3.2 seconds.

A Gilson HPLC system consisting of two 25 ml 306 Pump Heads, a 119Variable Dual Wavelength Detector, a 215 Liquid Handler, a 811C DynamicMixer, and a 806 Manometric Module, was used for product analysis andpurification.

Analytical HPLC on the individual components of the pyrazole libraryidentified, on average, the presence of pyrazole regioisomers. Theanalytical conditions used are as follows:

Column: Thomson Instrument Co. 50×4.6 mm C18 5 μm

Flow Rate: 1 ml/minutes.

Mobile Phase A: H₂O With 0.1% Trifluoroacetic Acid (TFA)

Mobile Phase B: Methanol (CH₃OH)

Gradient: 90%-10% mobile phase A in 12-minutes.

-   -   10%-90% mobile phase B in 12-minutes.

Wavelength: 254 nm

Injection: 10 μL

Analytical HPLC conditions were optimized for Preparative HPLC of thepyrazole compounds. The preparative conditions were as follows:

Column: Thomson Instrument Co. 50×21.5 mm C18 5 μm

Flow Rate: 11 ml/minutes.

Mobile Phase A: H₂O With 0.1% Trifluoroacetic Acid (TFA)

Mobile Phase B: Methanol (CH₃OH)

Gradient: 35%-10% mobile phase A in 7 minutes.

-   -   65%-90% mobile phase B in 7 minutes.

Wavelength: 254 nm

Injection: 250 μL

N-Alkylation of Pyrazole Amides

Amide nitrogen(s) of compounds of the present invention may be alkylatedusing the following procedure:

-   1. In a scintillation vial, the pyrazole starting material (1 eq)    was dissolved in DMF.-   2. Sodium hydride (15 eq) was placed in a vial fitted with a septum    and drying tube, and the vial was shaken for 1 hour.-   3. Alkylating reagent (e g., ethyl iodide) (15 eq) was then added,    and the reaction mixture was shaken for 5 hours.-   4. The reaction was then worked up by diluting the mixture with    ethyl acetate and washing with water and brine. The organic layers    were collected and dried over magnesium sulfate.-   5. The organic layers were filtered and concentrated in vacuo using    a Savant.-   6. Crude material was purified by flash chromatography using a    solvent system of 97:3 CH₂Cl₂:MeOH.

Example 2

This example shows the formation of the pyrazole ring having differentsubstituents. Referring to FIG. 7, the individual intermediates wereformed under the following reaction conditions.

-   2-Hydroxy-4-oxo-4-pyridin-3-ylbut-2-enoic acid methyl ester (1).    Acetyl pyridine (1.81 mL, 16.5 mmol) and methyl oxalate (3.12 g,    26.4 mmol) were dissolved in MeOH (30 mL, anhydrous). Sodium    methoxide (6.9 mL, 25% in MeOH) was added over 10 min. Reaction    solidified and was complete after 15 minutes. The solid mass was    dissolved when acidified with HCl (10%, aqueous). The pH was then    adjusted with NH₄OH (conc) until precipitation ceased. The resulting    solid was taken up in EtOAc. The aqueous layer was removed and    extracted twice with EtOAc. The combined EtOAc layers were washed    with water and brine, dried over MgSO₄, filtered, and concentrated    in vacuo. Collected 1 (2.90 g, 85%) as an off-white solid.-   1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carboxylic acid    methyl ester hydrochloride (2). To a solution of 1 (2.90 g, 14.0    mmol) in EtOH (60 mL, anhydrous) was added 3,4-dichlorophenyl    hydrazine hydrochloride (3.29 g, 15.4 mmol). The solution was heated    to reflux for 30 min and then cooled to 0° C. The precipitate was    collected by filtration and washed with H₂O and MeOH to yield 2    (3.79 g, 70%) as an off-white powder.-   1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carboxylic acid    methyl ester hydrochloride (3). A suspension of 2 (2.0 g, 5.74 mmol)    in THF (40 mL) and H₂O (11 mL) was treated with NaOH pellets (581    mg, 14.5 mmol) and heated to reflux for 1 h. The THF was removed in    vacuo and the pH of the remaining aqueous portion was adjusted to    1.5 with HCl (10%, aqueous). The resulting solid was dissolved in    EtOAc. The aqueous layer was removed and extracted with EtOAc-MeOH    (4:1). The combined organic layers were dried (brine and MgSO₄) and    concentrated in vacuo to yield 3 (1.61 g, 84%) as a white solid.-   1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazole-3-carbonyl azide    (4). A solution of 3 (100 mg, 0.27 mmol) and t-butyl alcohol (28.4    μL, 0.30 mmol) in DMF (5 mL, anhydrous) was cooled to 0° C.    Diphenylphosphoryl azide (64 μL, 0.30 mmol) was added to the    solution. Triethylamine (103 μL, 0.60 mmol) was then added over 10    min. The solution was stirred 1 h at 0° C. and allowed to warm to    room temperature and stir 16 h. The reaction was quenched with H₂O    and extracted with EtOAc. The combined organic layers were washed    with H₂O, dried (brine and MgSO₄), filtered, and concentrated in    vacuo. The resulting oil was purified by flash chromatography by    eluting with hexane-EtOAc (1:1). Collected 4 (86 mg, 90%) as a    yellow crystalline solid.-   [1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazol-3-yl]carbomic acid    tert-butyl (5). A solution of 4 (98 mg, 0.24 mmol) and t-butyl    alcohol (3 mL,) were heated to reflux for 4 h. The solution was    cooled and concentrated. The resulting oil was purified by flash    chromatography. Collected 4 (74 mg, 76%) as a clear oil.-   1-(3,4-Dichlorophenyl)-5-pyridin-3-yl-1H-pyrazol-3-yl amine (6). The    BOC-protected compound (5, 74 mg, 0.18 mmol) was dissolved in MeOH    (10 mL, anhydrous) and HCl (g) was bubbled through for 10 min. The    solution was stirred 3 h at room temperature. Concentrated in vacuo.    The remaining oil was dissolved in H₂O and neutralized with NaHCO₃    (sat. aqueous). The aqueous solution was extracted with CHCl₃; the    combined organic layers were dried (brine and MgSO₄), filtered, and    concentrated. The resulting oil was purified by flash chromatography    (chloroform-MeOH—NH₄OH 95:5:0.5) to yield 6 (39 mg, 70%) as a yellow    crystalline solid.

Example 3

This example demonstrates the synthesis of alkyl linking groups withincompounds of the present invention through the ethyl-methylamine linkinggroup example. Referring to FIG. 8 a, the individual intermediates wereformed under the following reaction conditions.

-   (2). Hydroxypyrazole 1 (7.0 g, 22.9 mmol) and POBr₃ (100 g, 348    mmol) were placed in a 115° C. oil bath with stirring. The slurry    was heated until just before all the hydroxypyrazole (1) totally    dissolved. The reaction was then cooled to RT and slowly poured into    EtOAc (250 mL, 0° C.). Crushed ice was slowly added to the EtOAc    solution. The pH of the aqueous layer was adjusted to 10 with NaOH    pellets. The biphasic solution was further diluted with water and    the organic layer was removed. The remaining aqueous layer was    extracted with EtOAc (×3) and the organic layers were combined,    dried (brine and MgSO₄), and concentrated in vacuo. The resulting    solid was purified by flash chromatography    (chloroform-methanol-NH₄OH 99.9:0.1:0.1 to 99.8:0.2:0.1) to afford    bromopyrazole 2 (3.44 g, 40%) as an off-white solid.-   (3). Bromopyrazole 2 (9.13 g, 24.7 mmol) was dissolved in THF (190    mL, anhydrous) and cooled to −78° C. n-BuLi (9.32 mL, 2.5 M in    hexane) was slowly added. The solution was stirred for 15 min at    −78° C. N-methyl formanilide (30.0 mL, 321 mmol) was slowly added to    the −78° C. solution, which was then allowed to slowly warm to RT    while stirring 16 h. The reaction was quenched with water and    extracted with EtOAc (×3). The combined organic layers were dried    (brine and MgSO₄) and concentrated in vacuo. The resulting solid was    purified by flash chromatography with a gradient of hexane-EtOAc    (2:1 to 1:1) to afford 3 (1.10 g, 14%) as an off-white solid.-   (4). β-Alanine ethyl ester HCl (279 mg, 2.52 mmol), 3 (400 mg, 1.26    mmol), and DIEA (437 μL, 2.52 mmol) were stirred in MeOH (4.0 mL,    anhydrous) along with molecular sieves for 16 h. The reaction    mixture was filtered and the filtrate was concentrated in vacuo.    Water was added to the resulting paste and the aqueous mixture was    extracted with EtOAc (×4); the combined EtOAc layers were dried    (brine and MgSO₄) and concentrated in vacuo. The resulting oil was    purified by flash chromatography by eluting with a gradient of EtOAc    to EtOAc-MeOH (10:1). The product 4 (180 mg, 35%) was isolated as a    white solid.-   (5). To a solution of pyrazole 4 (85 mg, 0.210 mmol) in THF (2 mL)    and water (2 mL) was added LiOH (10 mg). The resulting biphasic    mixture was stirred at RT for 4 h. The reaction was diluted with    water (4 mL) and the pH was adjusted to 6 with HCl (10% aqueous).    The cloudy solution was cooled to 5° C. for 16 h. The precipitate    was collected by filtration and then dried by concentrating in vacuo    with toluene. The acid 5 (70 mg, 85%) was isolated as a white solid.-   (6). To a solution of acid 5 (70 mg, 0.179 mmol) in DMF (6.0 mL,    anhydrous) was added DPPA (42.4 μL, 0.197 mmol) and DIEA (34.2 μL,    0.197 mmol). The resulting solution was then stirred at RT for 18 h.    L-Phe-NH₂ (147 mg, 0.895 mmol) was added and stirred at 60° C. for    8 h. The reaction was diluted with water and extracted with EtOAc    (×4). The combined EtOAc layers were washed with water (×2), dried    (brine and MgSO₄), and concentrated in vacuo. The resulting oil was    purified by flash chromatography with a gradient of    chloroform-MeOH—NH₄OH (98:2:0.5 to 95:5:0.5). The product 6 (40 mg,    41%) was isolated as a white foam.

Similarly, the phenyl linking group of the compounds of the presentinvention could be synthesized within this scheme using the boronic acidcoupling reaction. Referring to FIG. 8 b, the phenyl linking group canbe added to the pyrazole ring using the following synthetic methods.

-   (7). Bromopyrazole 2 (100 mg, 0.270 mmol), DME (2.33 mL), water (175    μL), Na₂CO₃ (142 mg, 1.34 mmol) and (4-aminocarbonyl phenyl)boronic    acid (97 mg, 0.588 mmol) were stirred together. Nitrogen was bubbled    through the reaction for 30 min. Pd(PPh₃)₄ (31 mg, 0.0268 mmol) was    added and the reaction was heated to 90° C. for 40 h. The reaction    was diluted with water and extracted with EtOAc (×3). The combined    organic layers were dried (brine and MgSO₄) and concentrated in    vacuo. The resulting oil was purified by flash chromatography    (CHCl₃-MeOH; 20:1). Product 7 (23 mg, 20%) was isolated as a solid.

Example 4

This example demonstrates the synthesis of prenylation inhibitors of thepresent invention with amine linking groups having different alkylsubstituents. Referring to FIG. 9, the individual intermediates wereformed under the following reaction conditions.

-   (8). N,N-Dimethylethylenediamine (39 μL, 0.35 mmol) and 3 (95 mg,    0.30 mmol) were dissolved in MeOH (20 mL, anhydrous) and stirred for    2 h at rt. NaBH₃CN (78 mg, 1.25 mmol) was added and stirred for 16 h    before removing the solvent in vacuo. The residue was taken up in    water and extracted with EtOAc. The organic layer was concentrated    in vacuo. The residue was purified by flash chromatography    (EtOAc-MeOH;4:1 with 1% NH₄OH) to give the product 8 (54 mg, 46%).-   (10). Ethyl acrylate (50 μL, 0.463 mmol), 8 (54 mg, 0.14 mmol), and    EtOH (10 mL, anhydrous) were added to a sealed tube. The reaction    was reacted under microwave conditions at 150° C. for 40 min. The    solvent was removed in vacuo. The residue was purified by flash    chromatography (EtOAc-MeOH; 7:3 with 1% NH₄OH). 9 was then subjected    to hydrolysis with KOH to give 10 (22 mg, 35%).-   (11). HOBt (14 mg, 0.10 mmol), 10 (22 mg, 0.05 mmol), L-Phe-NH₂    (16.4 mg, 0.10 mmol), EDCI (20 mg, 0.1 mmol) and DIEA (0.1 ml, 0.57    mmol) were stirred in CH₂Cl₂ (3.0 mL, anhydrous) for 16 h. The    reaction mixture-was washed with H₂O. The organic layer was washed    with brine and concentrated in vacuo. The crude reaction mixture was    purified by flash chromatography (EtOAc-MeOH; 7:3 with 1% NH₄OH) to    afford 11 (10 mg, 32%).

Example 5

This example demonstrates the synthesis of prenylation inhibitors of thepresent invention with different substituent on the pyrazole ring.Referring to FIG. 10, the individual intermediates were formed under thefollowing reaction conditions.

-   (12). NaOEt (21% w/v EtOH; 2.04 g, 30 mmol) was added to a dry flask    under N₂. The mixture was cooled to 0° C. During the cooling    process, ethyl nicotinate (4.53 g, 30 mmol) was added in one    portion. γ-Butyrolactone (2.58 g, 30 mmol) was added dropwise over    30 min. The mixture was stirred at 0° C. for 1 h. Upon removal of    the ice bath the reaction was allowed to warm to rt and was heated    to ˜65° C. overnight. The solvent was removed in vacuo and the    residue was diluted with H₂O, and extracted with diethyl ether to    remove any unreacted starting material. The aqueous phase was    acidified with 1N HCl and extracted with DCM. The organic layer was    washed with H₂O, brine, and dried (Na₂SO4), to yield 12 (2.87 g,    50%) as a brown oil.-   (13). To a solution of keto-lactone 12 (2.39 g, 12.5 mmol) in acetic    acid (100 mL) was added 3,4-dichlorophenylhydrazine HCl (2.93 g,    13.75 mmol) in one portion. The mixture was heated at reflux    overnight and then cooled to RT. The mixture was diluted with    de-ionized H₂O and extracted with EtOAc. The organic layer was    washed with saturated sodium bicarbonate (×2) and de-ionized H₂O    (×2), and dried over Na₂SO₄. Rotary evaporation and flash    chromatography afforded 13 as a light yellow solid (1.49 g, 34%).-   (14). To a solution of acetoxyethylpyrazole 13 (844 mg, 2.41 mmol)    in anhydrous DMF (20 mL) was added K₂CO₃ (501 mg, 3.62 mmol) and    ethyl-4-bromobutyrate (470 mg, 2.41 mmol). The reaction mixture was    heated to 80° C. and stirred overnight. The reaction mixture was    diluted with H₂O and extracted with EtOAc. The organic layer was    washed with H₂O, brine, dried (Na₂SO₄, and concentrated in vacuo.    The residue was purified by flash chromatography (hexane-EtOAc, 3:1)    to yield 14 (1.0 g, 82%) as a yellow oil.-   (15). Pyrazole 14 (1.00 g, 1.98 mmol) was dissolved in a mixture of    THF, MeOH, and 10% w/v NaOH solution (8:4:4). This solution was    stirred at rt overnight. The reaction mixture was concentrated in    vacuo and the residue was diluted with H₂O. The aqueous solution was    acidified to pH 4 with HCl (10% solution). The product was extracted    with 9:1 EtOAc/MeOH (×3). The combined organic layers were washed    with H₂O, dried (Na₂SO₄), and concentrated in vacuo to afford the    acid (803 mg, 1.84 mmol) which was dissolved in CH₂Cl₂ (25 mL). HOBt    (373 mg, 2.76 mmol) was added to the solution and was stirred for 20    minutes at rt. L-Phe-NH₂ (453 mg, 2.76 mmol), EDCI (707 mg, 3.68    mmol), and DIEA (641 μL, 3.68 mmol) were added and the solution was    stirred at rt overnight. The reaction mixture was washed with H₂O.    The organic layer was washed with brine and concentrated in vacuo.    Flash chromatography afforded impure 15 as a cream solid. The solid    was diluted with H₂O, filtered, and dried to yield 15 (651 mg, 61%)    as a white solid.

Example 6

This example demonstrates the synthesis of an ether linkage within theprenylation inhibitors of the present invention. Referring to FIG. 11,the ether linkage is formed on the pyrazole ring by the followingreactions.

-   (1). Sodium (38 g, 1.65 mmol) was slowly added to 900 mL of ethanol    and left to stir at RT for 4 h. Solvent was removed in vacuo. Ethyl    acetate (200 mL) was added to the sodium ethoxide followed by ethyl    nicotinate (22.6 mL, 0.166 mol) in 150 mL of ethyl acetate. The    reaction was heated to reflux for 16 h. Water (200 mL) was added to    the reaction mixture. The aqueous layer was acidified to pH 6 with    HCl and extracted with ether. Solvent was removed in vacuo to give a    brown oil which was purified by column chromatography (2:1,    hexanes-EtOAc), to give 1 (101 g, 52%).-   (2). Keto ester 1 (51.6 g, 267 mmol) and 3,4-dichloophenylhydrazine    hydrochloride (57.1 g, 267 mmol) were dissolved in 800 mL of acetic    acid and heated at 100 C. for 1 h. The reaction was cooled and left    to stand at RT overnight. The crystals that formed were filtered off    and washed with cold acetic acid to give 2 as an off-white solid (75    g, 92%).-   (3). Ethyl 4-bromobutyrate (28.8 g, 147.6 mmol), 2 (37.5 g, 123.0    mmol), K₂CO₃ (20.4 g, 147.6 mmol) were added to 1 L of DMF. The    reaction was heated at 70° C. for 16 h. Water was added, extracted    with ethyl acetate and dried over MgSO₄. Column chromatography (1:1    hexanes-EtOAc) gave 3 as a white solid (27.8 g, 54%).-   (4). NaOH (35 mL, 10% w/v) was added to 3 (2.2 g, 5.6 mmol) in    THF/MeOH (2:1, 90 mL) and heated at reflux for 2 h. The reaction    mixture was acidified to pH 6 with HCl. The solid that crashed out    of solution was filtered, washed with water and extracted with ethyl    acetate. The organic layers were concentrated in vacuo to give 4    (2.1 g, 95%).-   (5). To a solution of 4 (100 mg, 0.25 mmol) in CH₂Cl₂ (10 ml) were    added HOBt (69 mg, 0.5 mmol), EDCI (98 mg, 0.5 mmol),    L-phenylalaninamide (61 mg, 0.38 mmol), and DIEA (130 μl, 0.75 mmol)    sequentially. The reaction mixture was stirred at RT for 15 h. The    reaction mixture was diluted with CH₂Cl₂ (10 mL) and washed with    NaHCO₃ (5%, 10 mL) and brine (10 mL). The aqueous phase was    extracted with CH₂Cl₂ (3×10 mL). The organic phase was dried over    MgSO₄ and concentrated in vacuo to give a white solid. Column    chromatography (EtOAc) gave 5 as a white solid (94 mg, 70%).

Example 7

This example shows the synthesis of a prenylation inhibitor of thepresent invention having a central phenyl ring to which a phenyl linkinggroup is attached. Referring to FIG. 12, these phenyl groups areincorporated within the prenylation inhibitors of the present inventionby the following reactions.

-   (1). 1,3,5-Tribromobenzene (5.0 g, 15.9 mmol),    4-methoxycarbonylphenylboronic acid (5.71 g, 31.8 mmol), and cesium    carbonate (10.35 g, 31.8 mmol) were added to DME (20 mL). The flask    was evacuated and flushed with nitrogen three times. Pd(PPh₃)₄ was    added, the reaction vessel was covered with aluminum foil and the    reaction mixture was allowed to stir at RT for 16 h. The reaction    mixture was diluted with H₂O (50 mL), and extracted with EtOAc (3×40    mL). The extractions were combined, washed with brine, and dried    over MgSO₄. Concentration in vacuo, yielded a light brown solid.    Column chromatography (hexanes), yielded an off-white solid. (725    mg, 12%).-   (2). 3,4-Dichlorophenyl boronic acid (360 mg, 1.35 mmol), 1 (250 mg,    0.69 mmol), cesium carbonate (440 mg, 1.35 mmol), and H₂O (2 mL,)    were added to DME (10 mL,). The reaction vessel was evacuated and    flushed with nitrogen three times. Pd(PPh₃)₄ was added, the reaction    vessel was covered in aluminum foil and the reaction mixture was    allowed to stir at RT for 16 h. The reaction was diluted with H₂O    (50 mL), and extracted with EtOAc (3×40 mL). The extractions were    combined, washed with brine, and dried over MgSO₄. Concentration in    vacuo, yielded a light yellow solid. Column chromatography (99:1    hexanes-EtOAc) afforded a white solid, (98 mg, 33%).-   (3). Pyridine-3-boronic acid (46 mg, 0.38 mmol), 2 (98 mg, 0.19    mmol), cesium carbonate (124 mg, 0.38 mmol) and H₂O (0.4 mL), were    added to DME (2 mL). The reaction vessel was evacuated and flushed    with nitrogen three times. Pd(PPh₃)₄ was added and the reaction    vessel was covered in aluminum foil. The reaction mixture was heated    at 70° C. for 16 h. The reaction mixture was cooled to RT and    diluted with H₂O (50 mL), and extracted with EtOAc (3×40 mL). The    extractions were combined, washed with brine, and dried over MgSO₄.    Concentration in vacuo, yielded a brown solid. Column chromatography    (4:1 hexanes-EtOAc) afforded a light-brown solid, (9.3 mg, 11.3%).-   (4). Hydrolysis of 3 using conditions similar to those described    previously gave 4 which was used without further purification.-   (5). Coupling of 4 with Phe-NH₂ using the conditions previously    described afforded 5.

Example 8

This example shows the synthesis of a prenylation inhibitor of thepresent invention having a central phenyl ring attached to an etherlinking group. Referring to FIG. 13, this ether group is combined withthe central phenyl group by the following reactions.

-   (3). 3-4-Dichlorophenylboronic acid (3.0 g, 11.3 mmol),    3,5-dibromoanisole (2.0 g, 7.5 mmol), Cs₂CO₃ (4.9 g, 15.0 mmol) were    added to 60 mL of DME. A stream of nitrogen was gently bubbled    through the reaction mixture for 15 min to deaerate the reaction    mixture. Pd(PPh₃)₄ (868 mg, 0.752 mmol) was added and the reaction    mixture was reacted at RT for 16 h. The reaction mixture was diluted    with EtOAc (100 mL) and extracted with water (300 mL). The organic    phase was dried over MgSO₄ and concentrated in vacuo to give a dark    residue. Column chromatography afforded impure 3 as a white solid    (1.6 g).-   (4). 3-Pyridylboronic acid (1.18 g, 9.64 mmol), 3 (1.6 g, 4.82    mmol), Cs₂CO₃ (3.14 g, 9.64 mmol) were added to 40 mL of DME. A    stream of nitrogen was gently bubbled through the reaction mixture    for 15 min to deaerate the reaction mixture. Pd(PPh₃)₄ (557 mg,    0.482 mmol) was added and the reaction mixture was heated at reflux    for 16 h. The reaction mixture was diluted with EtOAc (30 mL) and    extracted with water (100 mL). The organic phase was dried over    MgSO₄ and concentrated in vacuo to give a dark residue. Column    chromatography afforded 4 as a white solid (390 mg, 25%).-   (5). 4 (50 mg, 0.152 mmol) was dissolved in HBr (aq. 48%, 100 μL)    and acetic acid (5 mL) and heated to 110° C. for 48 h. The reaction    mixture was diluted with water (20 mL) and basified to pH 7 with 10%    sodium hydroxide. The organic phase is concentrated down to a    residue and brought up in CH₂Cl₂ and filtered to give a gray solid    (41 mg, 86%).-   (6). To a solution of 5 (40 mg, 0.13 mmol) in DMF (5 mL) was added    K₂CO₃ (35 mg, 0.25 mmol) and ethyl-4-bromobutyrate (23 μL, 0.15    mmol). The solution was heated to 60° C. for 60 h. H₂O was added and    the product was extracted with CH₂Cl₂ (5×20 mL). The organic layer    was dried over MgSO₄ and solvent was removed in vacuo to give crude    6 as an oil. This was used without further purification.-   (7). Hydrolysis of 6 using conditions similar to those described    previously gave 7 that was used without further purification.-   (8). Coupling of 7 with Phe-NH₂ using the conditions previously    described afforded 8.

Example 9

This example shows the synthesis of a prenylation inhibitor of thepresent invention having a central pyrimidine ring matched with a phenyllinking group. Referring to FIG. 14, this phenyl group is combined withthe central pyrimidine group by the following reactions.

-   (1). 3-Acetyl pyridine (4.4 g, 40.0 mmol) was added to 150 mL of dry    CH₂Cl₂. TiCl₄ (1.0 M, 40.0 mL, 40.0 mmol) was added at 0° C.    followed by triethylamine (5.57 mL, 40.0 mmol). The reaction mixture    was stirred for 30 min before the dropwise addition of methy    4-formylbenzoate (5.0 g, 30.5 mmol, 50 mL CH₂Cl₂). The reaction    mixture was allowed to warm to RT and stirred for a further 16 h.    Solvent was removed in vacuo and the residue was washed with CH₂Cl₂    to give 1 as a yellow solid (5.0 g, 61%).-   (3). The chalcone 1 (500 mg, 1.87 mmol), 3,5-dichlorobenzamidine    hydrochloride 2 (422 mg, 1.87 mmol) and KOH (104 mg, 1.87 mmol) were    added to 10 mL of EtOH. The reaction mixture was heated at reflux    for 2 h. The reaction mixture was filtered and washed with EtOH (30    mL) and water (30 mL). This afforded a yellow solid 3 (281 mg, 34%)    which was dried in vacuo.-   (4). To a solution of 3 (130 mg, 0.30 mmol) in MeOH (10 mL) was    added NaOH (200 mg, 5.0 mmol). The solution was heated at reflux for    1 h. The reaction mixture was diluted with CH₂Cl₂/EtOH (3:1, 30 mL)    and acidified to pH 6 with 10% HCl. The aqueous phase was extracted    with CH₂Cl₂/EtOH (3:1, 3×30 mL). The organic phase was dried over    MgSO₄ and concentrated in vacuo to give a white solid (29 mg, 23%).-   (5). HOBt (9.3 mg, 76 μmol), 4 (29 mg, 69 μmol) were added to 5 mL    of CH₂Cl₂ and stirred for 10 min. EDCI (14.4 mg, 76 μmol),    L-phenylalaninamide (22.5 mg, 0.14 mmol), and DIEA (9.75 mg, 13.1    μl, 76 μmol) were added and the reaction mixture was stirred at RT    for 14 h. The reaction mixture was diluted with CH₂Cl₂/EtOH 3:1 (50    mL) and washed with NaHCO₃ (5%, 50 mL) and brine (50 mL). The    aqueous phase was extracted with CH₂Cl₂/EtOH (3:1, 3×50 mL). The    organic phase was dried over MgSO₄ and concentrated in vacuo to give    a white solid. The crude reaction mixture was purified by flash    chromatography (EtOAc→4:1 EtOAc-MeOH) to give 5 (6.7 mg, 17%).

Example 10

Similar to Example 9 above, this example shows the synthesis of aprenylation inhibitor of the present invention having a centralpyrimidine ring with an ether linking group. Referring to FIG. 15, thisether group is combined with the central pyrimidine group by thefollowing reactions.

-   (22). To a solution of ethyl nicotinoylacetate (1.58 g, 6.12 mmol)    and benzamidine hydrochloride (962 mg, 6.12 mmol) in EtOH (15 mL)    was added K₂CO₃ (1.69 g, 12.7 mmol). The reaction mixture was heated    to 70° C. for 16 h. The mixture was diluted with EtOAc and filtered    to remove K₂CO₃. The solid was washed repeatedly with EtOAc. The    organic layers were combined and the solvent was removed in vacuo.    The crude solid was washed with hexanes and used without further    purification.-   (23). Ethyl bromobutyrate (0.89 mL, 6.1 mmol), 22 (1.50 g, 6.1    mmol), and K₂CO₃ (1.27 g, 9.2 mmol) were combined in EtOH/DMF (4:1,    20 mL,). The reaction mixture was heated at 70° C. for 16 h, cooled    to RT, filtered to remove K₂CO₃ and concentrated in vacuo to give a    dark residue. Column chromatography gave 23 (740 mg, 33%).-   (24). To a solution of 23 (700 mg, 1.93 mmol) in THF/H₂O/MeOH 2:2:1    (20 mL) was added LiOH.H₂O (324 mg, 7.71 mmol). The solution was    heated at reflux for 1 h. The reaction mixture was concentrated in    vacuo and diluted with H₂O (15 mL). The solution was acidified to pH    ˜6 with c.HCl and extracted with CH₂Cl₂/EtOH (3:1, 3×20 mL). The    organic phase was dried over MgSO₄ and concentrated in vacuo to give    an orange-yellow solid (580 mg, 90%).-   (25). A solution of HOBt (94 mg, 61 μmol), EDCI (117 mg, 0.612    mmol), 24 (103 mg, 31 μmol) and DIEA (160 μL, 92 μmol) in CH₂Cl₂ (10    mL) was stirred at 0° C. for 30 min. L-phenylalaninamide (81 mg, 61    μmol) was added and the reaction mixture was stirred at RT for 16 h.    The reaction mixture was diluted with CH₂Cl₂/MeOH (3:1, 50 mL) and    washed with H₂O (50 mL). The aqueous phase was extracted with    CH₂Cl₂/MeOH (3:1, 3×50 mL). The organic phase was dried over MgSO₄    and concentrated in vacuo to give a light yellow solid. The crude    reaction mixture was washed repeatedly with CH₂Cl₂ to give 25 (75    mg, 51%).

Example 11

This example demonstrates the synthesis of a prenylation inhibitor ofthe present invention having a central oxazole ring and a phenyl linkinggroup. Referring to FIG. 16, this phenyl group is combined with thecentral oxazole group by the following reactions.

-   (15). To a solution of 3,4-dichlorophenacetyl bromide (2.67 g, 10.0    mmol) in CHCl₃ (40 mL) was added hexamethylenetetramine (1.4 g, 10.0    mmol). The reaction mixture was heated at 60° C. for 0.5 h. The    solid that formed was filtered and washed repeatedly with CHCl₃. The    white solid was then suspended in EtOH (50 mL). c.HCl (5 mL) was    added and the mixture was heated at reflux for 16 h. The mixture was    cooled in an ice-bath and the solid that formed was filtered and    washed with EtOH. The crude material (2.7 g) was used without    further purification.-   (16). Nicotinoyl chloride hydrochloride (1.56 g, 8.76 mmol) and 15    (1.96 g, 8.19 mmol) were suspended in pyridine (8 mL). The mixture    was heated at 100° C. for 2.5 h, cooled to RT and poured into H₂O    (20 mL). The orange solid thus formed was filtered, washed with H₂O    and dried in vacuo to give 16 (1.18 g, 50%).-   (17). To a solution of 16 (1.02 g, 3.32 mmol) in acetic anhydride    (10 mL) was added phosphoric acid (85%, 850 μl). The brown solution    was heated at reflux for 3 h. The reaction mixture was reduced to a    residue under reduced pressure, redissolved in CH₂Cl₂/EtOH 3:1 and    extracted with NaHCO₃ (5%). The organic phase was dried over MgSO₄    and concentrated in vacuo. Column chromatography afforded 17 (338    mg, 35%).-   (18). To a solution of 17 (170 mg, 0.58 mmol) in CHCl₃ (3 mL) was    added bromine (90 μl, 280 mg, 1.75 mmol). The mixture was subject to    microwave heating for 20 min (CEM Explorer, power 200 W, temperature    105° C., pressure 100 PSI). The reaction mixture was dried down    under reduced pressure to give 18 as a yellow solid (185 mg, 86%).-   (19). 4-Methoxycarbonylphenylboronic acid (188 mg, 1.03 mmol), 18    (189 mg, 0.51 mmol), Cs₂CO₃ (667 mg, 2.04 mmol) were added to 15 mL    of DME and 2 mL, of H₂O. A stream of nitrogen was gently bubbled    through the reaction mixture for 15 min to deaerate the reaction    mixture. Pd(PPh₃)₄ (15 mg, 13 μmol) was added and the reaction    mixture was heated at reflux for 16 h. The reaction mixture was    diluted with CH₂Cl₂ (50 mL) and extracted with NaHCO₃ (50 mL, 5%).    The organic phase was dried over MgSO₄ and concentrated in vacuo to    give a dark residue. Column chromatography afforded 19 as a white    solid (60 mg, 27%).-   (20). To a solution of 19 (68 mg, 0.16 mmol) in MeOH/CH₂Cl₂ (4:1, 10    mL) was added NaOH (128 mg, 3.2 mmol). The yellow solution was    heated at reflux for 1 h. The reaction mixture was diluted with    MeOH/CH₂Cl₂ (1:1, 50 mL) and acidified to pH 6 with 5% HCl. The    aqueous phase was extracted with CH₂Cl₂/MeOH (3:1, 3×30 mL). The    organic phase was dried over MgSO₄ and concentrated in vacuo to give    a white solid (67 mg, 100%).-   (21). To a solution of 20 (67 mg, 0.16 mmol) in CH₂Cl₂ (10 ml) were    added HOBt (49 mg, 0.32 mmol), EDCI (61 mg, 0.32 mmol),    L-phenylalaninamide (52 mg, 0.32 mmol), and DIEA (41 mg, 56 μl, 0.32    mmol) sequentially. The reaction mixture was stirred at RT for 15 h.    The reaction mixture was diluted with CH₂Cl₂/EtOH 3:1 (50 mL) and    washed with NaHCO₃ (5%, 50 mL) and brine (50 mL). The aqueous phase    was extracted with CH₂Cl₂/EtOH 3:1 (3×50 mL). The organic phase was    dried over MgSO₄ and concentrated in vacuo to give a white solid.    The crude reaction mixture was washed with hexane and MeOH to give    21 (45 mg, 50%).

Example 12

This example demonstrates the synthesis of a prenylation inhibitor ofthe present invention having a central pyrazole ring and a thiophenelinking group. Referring to FIG. 17, this thiophene group is combinedwith the central pyrazole group by the following reactions.

-   (1). 3,5-Thiophenedicarboxylic acid (21.2 g, 123 mmol) was dissolved    in methanol (70 mL, anhydrous) and dichloroethane (70 mL,    anhydrous). c.H₂SO₄ (10.6 mL) was added and the reaction mixture was    heated at reflux for 40 h. Solvent was removed in vacuo and the    solid was suspended in EtOAc, washed with saturated NaHCO₃ (2×200    mL) and brine and dried over MgSO₄. Solvent was removed in vacuo to    yield 1 as a white powder (22.4 g, 91%).-   (2). To NaH (7.68 g, 192 mmol, 60% dispersion in oil) was added    acetyl pyridine (14.1 mL, 128 mmol) dissolved in THF (360 mL,    anhydrous). The reaction mixture was heated at reflux for 1 h,    cooled to RT and 1 (22.4 g, 112 mmol) was added over 30 min. The    reaction was allowed to stir at RT for 48 h. Saturated NH₄Cl (100    mL) was added and concentrated under vacuum to give a solid. The    solid was washed with H₂O and EtOAc and dried under vacuum at 60° C.    The solid was then suspended in boiling methanol and filtered to    give 2 as brown solid (15.4 g, 28%).-   (3). 3,4-Dichlorophenylhydrazine hydrochloride (11.6 g, 54.4 mmol),    and 2 (15.4 g, 49.4 mmol) were suspended in methanol (450 mL,    anhydrous) and heated at reflux for 20 h. The solvent was removed in    vacuo, the residue was dissolved in NaHCO₃, extracted with EtOAc    (3×150 mL) and dried over MgSO₄. Column chromatography (2:1    hexanes-EtOAc to EtOAc) gave 3 (10.6 g, 59%) as a yellow solid.-   (4). Methyl ester 3 (156 mg, 0.36 mmol) was dissolved in THF (3 mL)    and H₂O (3 mL). LiOH (17 mg, 7.1 mmol) was added and the solution    was stirred at RT for 20 h. Solvent was removed in vacuo and the    residue was dissolved in H₂O. The aqueous layer was washed with    CHCl₃ and then acidified with HCl (10% solution). The solid that    precipitated out was collected, washed with H₂O and dried to give 4    (70 mg, 46%).-   (5). To a solution of 4 (70 mg, 0.17 mmol) in CH₂Cl₂ (3 mL) were    added DMAP (catalytic amount), EDCI (48 mg, 0.25 mmol), and    L-phenylalaninamide (72 mg, 0.44 mmol). The reaction mixture was    stirred at RT for 16 h. The reaction mixture was diluted with CH₂Cl₂    (10 mL) and washed with NaHCO₃ (5%, 10 mL) and brine (10 mL). The    aqueous phase was extracted with CH₂Cl₂ (3×10 mL). The organic phase    was dried over MgSO₄ and concentrated in vacuo to give a white    solid. Column chromatography (EtOAc) gave 5 as a white solid (33 mg,    35%).

Example 13

This example demonstrates the synthesis of a prenylation inhibitor ofthe present invention having a central pyrazole ring and an aminelinking group. Referring to FIG. 18, this amine group is combined withthe central pyrazole group by the following reactions.

-   (1). Sodium (38 g, 1.65 mmol) was slowly added to 900 mL of ethanol    and left to stir at RT for 4 h. Solvent was removed in vacuo. Ethyl    acetate (200 mL) was added to the sodium ethoxide followed by ethyl    nicotinate (22.6 mL, 0.166 mol) in 150 mL of ethyl acetate. The    reaction was heated to reflux for 16 h. Water (200 mL) was added to    the reaction mixture. The aqueous layer was acidified to pH 6 with    HCl and extracted with ether. Solvent was removed in vacuo to give a    brown oil which was purified by column chromatography (2:1,    hexanes-EtOAc), to give 1 (16.7 g, 52%).-   (2). Keto ester 1 (51.6 g, 267 mmol) and 3,4-dichloophenylhydrazine    hydrochloride (57.1 g, 267 mmol) were dissolved in 800 mL of acetic    acid and heated at 100 C for 1 h. The reaction was cooled and left    to stand at RT overnight. The crystals that formed were filtered off    and washed with cold acetic acid to give 2 as an off-white solid (75    g, 92%).-   (3). Hydroxypyrazole 2 (1.78 g, 5.8 mmol) and POBr₃ (23.4 g, 81.5    mmol) were placed in a 115° C. oil bath with stirring. The slurry    was heated until just before all of 2 totally dissolved. The    reaction was then cooled to RT and slowly poured into EtOAc (100 mL,    0° C.). Crushed ice was slowly added to the EtOAc solution. The pH    of the aqueous layer was adjusted to 10 with NaOH pellets. The    biphasic solution was further diluted with water and the organic    layer was removed. The remaining aqueous layer was extracted with    EtOAc (×3) and the organic layers were combined, dried (brine and    MgSO₄), and concentrated in vacuo. The resulting solid was purified    by flash chromatography (chloroform-methanol-NH₄OH 99.9:0.1:0.1 to    99.8:0.2:0.1) to afford bromopyrazole 3 (1.28 g, 59%) as an    off-white solid.-   (4). CuI (12 mg, 63 μmol), N,N-diethylsalicylamide (40 mg, 0.2    mmol), 5-bromo-1-(3,4-dichlorophenyl)-3-pyridylpyrazole (250 mg,    0.68 mmol), and potassium phosphate (288 mg, 1.36 mmol) were added    to an oven dried round-bottomed flask. The flask was evacuated and    back-filled with nitrogen three times. A solution of 2-pyrrolidinone    (170 mg, 2.0 mmol) in DMF (5 mL, anhydrous) was added via syringe at    RT. The reaction was heated at 100° C. for 48 h. Solvent was removed    in vacuo and the residue was partitioned between ethyl acetate and    water. The organic layer was washed with brine, and dried over    MgSO₄. Column Chromatography afforded 4 as an oily white solid (75    mg, 33% yield).-   (5). Pyrazole 4 (165 mg, 0.44 mmol) was dissolved in methanol (10    mL). KOH (560 mg, 10.0 mmol) in H₂O (2 mL) was added and the    reaction mixture was stirred at 60° C. for 4 h. Solvent was removed    in vacuo and the residue was partitioned between EtOAc and H₂O. The    aqueous layer was acidified with acetic acid to pH 5 and extracted    with EtOAc. The organic layer was washed with brine and dried over    MgSO₄. Solvent was removed in vacuo to yield 5 (100 mg, 59%).-   (6). To a solution of 5 (19 mg, 0.05 mmol) in CH₂Cl₂ (3 mL) were    added HOBt (14 mg, 0.1 mmol), EDCI (19 mg, 0.1 mmol),    L-phenylalaninamide (16 mg, 0.1 mmol), and DIEA (50 μl, 0.3 mmol).    The reaction mixture was stirred at RT for 16 h. The reaction    mixture was diluted with CH₂Cl₂ (10 mL) and washed with brine (5    mL). The aqueous phase was extracted with CH₂Cl₂ (3×10 mL). The    organic phase was dried over MgSO₄ and concentrated in vacuo. Column    chromatography (9:1, EtOAc-MeOH) gave 6 as a yellow solid (21 mg,    78%).

Example 14

This example illustrates a method for preparing and purifying GGPTase I.

GGPTase I was prepared and purified according to the method described byZhang et al., J. Biol. Chem., 1994, 9, 23465-23470, which isincorporated herein in its entirety by this reference.

Production of Recombinant Virus

Sf9 cells were obtained from the American Tissue Culture Collection. Thecells were maintained in Grace's medium (Gibco), supplemented with about3.3 mg/ml lactalbumin hydrolystate (Difco), about 3.3 mg/ml yeastolate(Difco), about 10% (v/v) fetal bovine serum (HyClone Laboratories,Logan, Utah), antibiotic-antimycotic mixture (Gibco), and about 0.1%Pluronic F-68 (Gibco) in 125 ml Spinner flask (available from Techne,Princeton, N.J.). To generate recombinant baculovirus, about 2×10⁶ Sf9cells were transfected with about 0.5 μg of BaculoGold wild-type viralDNA (available from PharMingen) and about 2 μg of either pVL-Fα (for αsubunit expression) or pVL-Gβ (for GGPTase-Iβ subunit expression) usingcalcium-phosphate precipitation according to the manufacturer'sinstructions (PharMingen). The virus from each transfection washarvested after about 4 days and screened using a plaque assay asdescribed by Summers and Smith, A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures, Texas AgriculturalExperimentation Station, Bulletin #1555 (1987). Recombinant virusesobtained from this screen were subjected to two further rounds of plaqueamplification to obtain purified viruses.

Production and Purification of Recombinant GGPTase-I

The purified recombinant viruses containing the cDNA sequences for the αsubunit of FPTase and GGPTase, and the β subunit of GGPTase-I were usedto co-infect about 1.5×10⁶ Sf9 cells at multiplicities of infection of5. Cells were harvested at about 65 hours post-infection bycentrifugation at about 800×g for about 15 minutes. The cells werewashed once with phosphate-buffered saline and the resulting cell pelletflash-frozen in liquid nitrogen. Cell extracts were prepared by thawingthe cell suspension in 5 volumes of about 20 mM Tris-HCl, pH 7.7, about1 mM EDTA, 1 mM EGTA, about 1 mM and a protease inhibitor mixture(Moomaw et al., Methods Enzymol., 1995, 250, 12-21), incubating the cellsuspension oil ice for about one hour, and disrupting using six strokesof a Dounce homogenizer. The resulting extract was centrifuged for about1 hour at about 30,000×g, and the supernatant (designated as the solubleextract) was fractionated on a 5.0×10.0 cm column of DEAE-Sephacel(available from Pharmacia). The DEAE-Sephacel was first equilibratedwith 50 mM Tris-Cl, pH 7.7, 1 mM DTT (Buffer A) at 4° C. The solubleextract containing about 160 mg protein was loaded into the DEAE column,which was then washed with about 50 ml Buffer A and eluted with a 200 mlgradient of 0-500 mM NaCl in Buffer A. Fractions of 3 ml were collected.The fractions containing the peak of GGPTase-I activity were pooled,concentrated and exchanged into Buffer A, and then loaded into a Q-HPcolumn (1.0×20 cm, available from Pharmacia). The column was washed withabout 20 ml of buffer A and eluted with a 200 ml gradient of 0-500 mMNaCl in Buffer A. The peak fractions, containing essentially homogeneousGGPTase-I, were pooled, flash-frozen in aliquots and stored at −80° C.

Example 15

This example illustrates a method for determining GGPTase-I activity.

GGPTase-I activity was determined by the method of Casey et al, Proc.Natl. Acad. Sci. USA, 1991, 88, 8631-8635. This method measures thetransfer of isoprenoid from ³H-geranylgeranyl diphosphate (GGPP) into aRas protein with a C-terminal leucine-for-serine substitution(designated as Ras-CVLL).

Example 16

This example illustrates GGPTase I and FPTase inhibitory activities ofsome of the compounds of the present invention.

Assays for the inhibition studies of GGPTase I were performed in amanner analogous to that described by Casey, et al., Proc. Natl. AcadSci. USA, 1991, 88, 8631-8635, with the following modifications. Forthose assays, the reaction mixtures contained the following componentsin 50 μl:0.25 μM [³H]GGPP (sp. act. 8-10 Ci/mmol), 2.5 μM Ras-CVLL, 50mM Tris-Cl, pH 7.7, 20 mM KCI, 5 mM MgCl₂, 5 μM ZnCl₂, 1 mM DTT, 0.5 mMZwittergent 3-14 and the desired amount of the compound to be tested forinhibitory potential. After pre-equilibrating the assay mixture at 30°C. in the absence of the enzyme, the reaction was initiated by additionof the enzyme (75 ng). Following an about 10 minute incubation at about30° C., the reactions were terminated by addition of about 0.5 ml ofabout 4% SDS. About 40 mg of bovine brain membranes was added to thesamples to enhance recovery during precipitation. Product wasprecipitated by addition of about 0.5 ml of 30% TCA, allowed to stand atroom temperature for about 15 minutes, and processed by filtrationthrough glass-fiber filters as described previously (Reiss et al.,Methods: Companion to Methods in Enzymology, 1991, 1, 241-245).Reactions were never allowed to proceed to more than 10% completionbased on the limiting substrate. Assays for the inhibition studies ofFPTase were performed analogous the GGPTase I inhibition studies, except[³H]GGPP was replaced with 0.25 μM of [³H]FPP (sp. act. 8-10 ci/mmol)and Ras-CVLL, was replaced with 1 μM H-Ras.

Using the method described above, the GGPTase I inhibitory activities ofsome of the compounds of the present invention were evaluated. Compoundsof Table 1 were found to have inhibitory activity. Mixtures ofregioisomers and/or enantiomers are used unless indicated otherwise (forexample, the position of substituents on a cyclic or heterocyclic moietywithin the backbone of the compounds of the present invention).

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A compound of the formula:

or a pharmaceutically-acceptable salt thereof, wherein R₁ is phenyl,benzyl, methyl, ethyl, propyl, 3,4-dimethylphenyl, 3,4-difluorophenyl,3,4-dichlorophenyl, 3,5-dichlorophenyl, CH₂CF₃, 4-trifluoromethylphenyl,4-nitrophenyl, 4-bromophenyl, 3-bromophenyl, 4-methylphenyl,4-methoxyphenyl, 4-chloro-2-methylphenyl, 4-fluorophenyl,4-sulfonamidophenyl, 3-methoxyphenyl, 4-chlorophenyl, 3-chlorophenyl,3,5-difluorophenyl, 4-aminophenyl, ethanol, or 3,4-methylenedioxyphenyl;R₂ is pyridine; R₃ is H, CH₂CH₂OH, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂,CH₂CH₂NHCH₃, CH₂OH, (CH₂)₃OH, CH₂CH₂CO₂H, CH₂CO₂H, CH₂CH₂SOCH₃,CH₂CH₂SO₂CH₃, CH₂CH₂SH or CH₂CH₂SCH₃; R₄ is H, NH₂, CON(CH₃)₂, CO₂H, CN,CH₂OH, CONH₂, CSNH₂, CONHOH, C(NH)NH₂, CONHNH₂, CONHCH₃, CH₂OCH₃,CONH-cyclohexyl, CO₂CH₃, or

R₅ is isopropyl, benzyl, 4-trifluoromethylbenzyl, 4-cyanobenzyl,4-benzoylbenzyl, 3-chlorobenzyl, pentafluorobenzyl, 3,4-dichlorobenzyl,2-fluorobenzyl, 4-methoxybenzyl, CH₂CH₂-phenyl, 4-fluorobenzyl,4-phenylbenzyl, CH₂COOH, CH₂CH₂COOH, (CH₂)₄NH₂, CH₂CH₂SCH₃,4-hydroxybenzyl, CH₂-naphthyl, 4-methylbenzyl, CH₂-cyclohexane,4-chlorobenzyl, phenyl, 2-hydroxybenzyl, 4-tertbutoxybenzyl,4-aminobenzyl, CH₂OH, (CH₂)₃NHC(NH)NH₂ or CH₂CH(CH₃)₂; and, R₆ is H,methyl, ethyl, propyl, isopropyl, CH₂CO₂H, CH₂CO₂Et, benzyl, orCH₂-(2-methoxynaphthyl); or, R₅ and R₆ together form:


2. A pharmaceutical composition comprising a compound of claim 1 or apharmaceutically-acceptable salt thereof, and a pharmaceutically-acceptable carrier.